Methods for the treatment of il-1beta related conditions

ABSTRACT

Disclosed are methods and materials for inhibiting (e.g., treating or preventing) uveitis in a subject, including treatment refractory uveitis, using anti-IL-1β binding molecules (e.g., IL-1β binding antibodies or binding fragment thereof).

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/444,638 filed Feb. 18, 2011, U.S. Provisional Application No.61/334,125 filed May 12, 2010, and U.S. Provisional Application No.61/332,658 filed May 7, 2010, the disclosures of which are incorporatedby reference herein in their entirety.

FIELD OF INVENTION

The present disclosure relates to methods and materials for treating orpreventing uveitis in a subject, including treatment refractory uveitis.

BACKGROUND OF THE INVENTION

Uveitis generally refers to intraocular inflammation and may, forexample, affect the anterior portion of the uvea and/or the posteriorportion of the uvea. Uveitis is a prevalent cause of visual impairmentin many countries. The anterior portion of the uvea includes the irisand ciliary body. The posterior portion of the uvea includes thechoroid. In addition to providing most of the blood supply of theintraocular structures, the uveal coat acts as a conduit for immunecells, particularly lymphocytes, to enter the eye. Consequently, it isdirectly involved in many intraocular inflammatory processes.

The international Uveitis Study Group classifies uveitis in terms of theeye(s) involved (i.e., unilateral or bilateral), course (i.e., acute,lasting less than 12 weeks, or chronic, lasting more than 12 weeks), andanatomical location in the eye (Bloch-Michel et al., Am J Ophthalmol.,103:234-235, 1987). Further standardization of the characterization andnomenclature of uveitis is provided by the SUN working group (Jabs, etal., Am J Ophtalmol., 140:509-516, 2005). Anterior uveitis includes, forexample, iritis, anterior eyelids, and iridocyclitis involving the irisand/or pars plicata (anterior ciliary body). Intermediate uveitisincludes, for example, pars planitis, posterior cyclitis, hyalitis, andbasal retinochoroiditis, referring to inflammation of the pars plana(posterior ciliary body) and/or adjacent peripheral retina. Posterioruveitis includes focal, multifocal, or diffuse choroiditis; retinitis;neuroretinitis, retinochoroiditis; and chorioretinitis; the latter 2terms indicate which tissue appears primarily involved. Panuveitisrefers to inflammation that involves both the anterior and posteriorsegments. Uveitis may be further classified on the presence or absenceof granulomatous inflammation, marked by “mutton fat” keraticprecipitates, iris nodules, and/or choroidal granulomas.

Estimates indicate that uveitis may account for about 10% of the visualhandicaps in the western world (Nussenblatt, Int Ophthalmol.,14:303-308, 1990) and up to 15% of all cases of total blindness in theUnited States (Rothova el al., Br J Ophthalmol., 80:332-436, 1996).Legal blindness develops in at least one eye in 22% of all uveitispatients and in about 23% of all who require intraocular surgery. Inaddition, visual acuity loss to worse than 6/18 in at least one eyeoccurs in 35% of patients with uveitis, mainly as a result of persistentmacular edema (Rothova et al., ibid). The ocular complications ofuveitis are usually involved in the decrease in visual acuity.

IL-1β is a proinflammatory cytokine secreted by a number of differentceil types including monocytes and macrophages. When released as part ofart inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotropin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types. Theinterleukin-1 (IL-1) family of cytokines has been implicated in a numberof disease states. IL-1 family members include IL-1α, IL-1β, and IL-1Ra.Although related by their ability to bind to IL-1 receptors (IL-1R1 andIL-1R2), each of these cytokines is different, being expressed by adifferent gene and having a different primary amino acid sequence.Furthermore, the physiological activities of these cytokines can hedistinguished from each other.

Effective treatment of uveitis, including an acute uveitis exacerbation(e.g., uveitis flare, uveitis attack), with a complete resolution ofinflammatory findings, is important for a better visual outcome. Thelonger a uveitis exacerbation goes unresolved, the greater are thechances of more severe sequela, incomplete resolution, and/or loss ofvision. There remains a need for effective methods of treating andpreventing uveitis. Including treatment of refractory uveitis andprevention of uveitis exacerbations including in at risk subjects.

SUMMARY OF THE INVENTION

The present disclosure relates to materials and methods for inhibiting(e.g., treating or preventing) uveitis in a subject, comprisingadministering to the subject an effective amount of anti-IL-1β antibodyor binding fragment thereof. Surprisingly, the methods disclosed hereinprovide an effective means for inhibiting (e.g., treating or preventing)treatment refractory (e.g., treatment resistant) uveitis, with orwithout the use of additional pharmaceutical compositions, such as forexample a non-steroid immunosuppressant, a non-steroid anti-inflammatoryand/or a steroid. Such materials and methods may be used to treat amammalian (e.g., human) subject suffering from uveitis disease (e.g.,treatment refractory uveitis) or to prevent occurrence or reduce thefrequency and/or severity of same in an at risk subject.

The present disclosure provides a method of inhibiting uveitis in asubject, the method comprising administering to the subject an effectiveamount of anti-IL-1β antibody or binding, fragment thereof, wherein theuveitis is treatment refractory (e.g., treatment resistant) uveitis. Insome embodiments, the method of inhibiting uveitis in a subject is amethod of preventing uveitis in the subject. In some embodiments, themethod of inhibiting uveitis in a subject is a method of treatinguveitis in the subject.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject is inhibiting an acute uveitisexacerbation.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject increases in the interval between acuteuveitis exacerbations.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject decreases the frequency of acute uveitisexacerbations.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject decreases the likelihood of experiencingan acute uveitis exacerbation.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject prevents an acute uveitis exacerbation.

In some embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject treats an acute uveitis exacerbation. Insome embodiments of each or any of the aforementioned methods,inhibiting uveitis in a subject decreases the severity of an acuteuveitis exacerbation.

In some embodiments of each or any of the aforementioned methods, thetreatment refractory uveitis is uveitis that is refractory to treatmentwith a pharmaceutical composition comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid. In someembodiments, the non-steroid immunosuppressant is a DNA synthesisinhibitor, a cyclosporine, a mycophenolate or a colchicine. In someembodiments, the DNA synthesis inhibitor is azathioprine, an alkylatingagent, an anti-metabolite (e.g., methotrexate), X-ray therapy,chlorambucil or cyclophosphamide. In some embodiments, the non-steroidanti-inflammatory is a TNF inhibitor, an IL-6 inhibitor or an IL-17inhibitor. In some embodiments, the steroid is a steroid hormoneselected from the group consisting of prednisone, methylprenisolone,prednisolone, a cortisol, an andrenocorticotrophic hormone and aglucocorticoid (e.g., dexamethasone).

In some embodiments of each or any of the aforementioned methods, thesubject is receiving concurrently for the inhibition of said uveitis atleast one (e.g., one or two) pharmaceutical compositions comprising anon-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid. In some embodiments, the subject is receiving concurrently forthe inhibition of said uveitis one pharmaceutical composition comprisinga non-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid.

In some embodiments of each or any of the aforementioned methods, thesubject is not receiving concurrently for the inhibition of said uveitisa pharmaceutical composition selected from the group consisting of apharmaceutical composition comprising a non-steroid immunosuppressant, apharmaceutical composition comprising a non-steroid anti-inflammatoryand a pharmaceutical composition comprising a steroid. In someembodiments, the subject is not receiving concurrently for the treatmentor prevention of said uveitis a pharmaceutical composition comprising anon-steroid immunosuppressant. In some embodiments, the subject is notreceiving concurrently for the treatment or prevention of said uveitis apharmaceutical composition comprising a non-steroid anti-inflammatory.

In some embodiments of each or any of the aforementioned methods, thesubject has received prior treatment for uveitis with one or morepharmaceutical compositions comprising a non-steroid immunosuppressant,a non-steroid anti-inflammatory or a steroid. In some embodiments, thesubject had an adverse reaction or hypersensitivity to said priortreatment of uveitis with one or more pharmaceutical compositionscomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid. In some embodiments, the subject failedsaid prior treatment of uveitis with one or more pharmaceuticalcompositions comprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid.

In some embodiments of each or any of the aforementioned methods, thenon-steroid immunosuppressant is a DNA synthesis inhibitor, acyclosporine, a mycophenolate or a colchicine. In some embodiments, theDNA synthesis inhibitor is azathioprine, an alkylating agent, ananti-metabolite (e.g., methotrexate). X-ray therapy, chlorambucil orcyclophosphamide. In some embodiments, the non-steroid anti-inflammatoryis a TNF inhibitor, an IL-6 inhibitor or an IL-17 inhibitor. In someembodiments, the steroid is a steroid hormone selected from the groupconsisting of prednisone, methylprenisolone, prednisolone, a cortisol,an andrenocorticotrophic hormone and a glucocorticoid (e.g.,dexamethasone).

In some embodiments of each or any of the aforementioned methods, thesubject is receiving concurrently for the treatment or prevention ofsaid uveitis at least one pharmaceutical composition comprising anon-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid, and wherein said method provides a reduction in the dosage ofsaid at least one pharmaceutical composition. In some embodiments, thereduction in the dosage is a reduction in the dose of said at least onepharmaceutical composition, as compared to the dose prior toadministering the anti-IL-1β antibody or binding fragment thereof. Insome embodiments, the reduction in the dosage is a reduction in thefrequency of doses of said at least one pharmaceutical composition, ascompared to the frequency of doses prior to administering the anti-IL-1βantibody or binding fragment thereof. In some embodiments, the dosage ofa pharmaceutical composition comprising a non-steroid immunosuppressantis reduced. In some embodiments, the non-steroid immunosuppressant is aDNA synthesis inhibitor, a cyclosporine, mycophenolate or a colchicine.In some embodiments, the DNA synthesis inhibitor is azathioprine ormethotrexate. In some embodiments, the dosage of a pharmaceuticalcomposition comprising a steroid is reduced. In some embodiments, thesteroid is a steroid hormone selected from the group consisting ofprednisone, prednisolone, methylprednisolone, a cortisol, anandrenocorticotrophic hormone and a glucocorticoid.

In some embodiments of each or any of the aforementioned methods, themethod is a method of inhibiting an acute uveitis exacerbation in asubject diagnosed with uveitis, and wherein the acute uveitisexacerbation has a severity grade of at least a 2 step increase inintraocular inflammation according to SUN criteria.

The present disclosure provides a method of treating uveitis in asubject, the method comprising administering to the subject an effectiveamount of anti-IL-1β antibody or binding fragment thereof, wherein theuveitis is treatment refractory (e.g., treatment resistant) uveitis. Insome embodiments, the treatment refractory uveitis is uveitis that isrefractory to treatment with a pharmaceutical composition comprising anon-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid. In some embodiments, the non-steroid immunosuppressant is a DNAsynthesis inhibitor, a cyclosporine, a mycophenolate or a colchicine. Insome embodiments, the DNA synthesis inhibitor is azathioprine, analkylating agent, an anti-metabolite (e.g., methotrexate), X-raytherapy, chlorambucil or cyclophosphamide. In some embodiments, thenon-steroid anti-inflammatory is a TNF inhibitor, an IL-6 inhibitor oran IL-17 inhibitor. In some embodiments, the steroid is a steroidhormone selected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid.

The present disclosure also provides a method of treating or preventinguveitis in a subject, the method comprising administering to the subjectan effective amount of anti-IL-1β antibody or binding fragment thereof,wherein the subject is receiving concurrently for the treatment orprevention of said uveitis at least one (e.g., one or two)pharmaceutical compositions comprising a non-steroid immunosuppressant,a non-steroid anti-inflammatory or a steroid. In some embodiments, thesubject is receiving concurrently for the treatment or prevention saiduveitis one pharmaceutical composition comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid. In someembodiments, the non-steroid immunosuppressant is a DNA synthesisinhibitor, a cyclosporine, a mycophenolate or a colchicine. In someembodiments, the DNA synthesis inhibitor is azathioprine, an alkylatingagent, an anti-metabolite (e.g., methotrexate), X-ray therapy,chlorambucil or cyclophosphamide. In some embodiments, the non-steroidanti-inflammatory is a TNF inhibitor, an IL-6 inhibitor or an IL-17inhibitor. In some embodiments, the steroid is a steroid hormoneselected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid.

The present disclosure also provides a method of treating or preventinguveitis in a subject, the method comprising administering to the subjectan effective amount of anti-IL-1β antibody or binding fragment thereof,wherein the subject is receiving concurrently for the treatment orprevention of said uveitis a pharmaceutical composition comprising aninterferon (e.g., IFN-α)

The present disclosure also provides a method of treating or preventinguveitis in a subject, the method comprising administering to the subjectan effective amount of anti-IL-1β antibody or binding fragment thereof,wherein the uveitis is treatment refractory (e.g., treatment resistant)uveitis and wherein the subject is hot receiving concurrently for thetreatment or prevention of said uveitis a pharmaceutical compositionselected from the group consisting of a pharmaceutical compositioncomprising a non-steroid immunosuppressant, a pharmaceutical compositioncomprising a non-steroid anti-inflammatory and a pharmaceuticalcomposition comprising a steroid. In some embodiments, the subject isnot receiving concurrently for the treatment or prevention of saiduveitis a pharmaceutical composition comprising a non-steroidimmunosuppressant. In some embodiments, the subject is not receivingconcurrently for the treatment or prevention of said uveitis apharmaceutical composition comprising a non-steroid anti-inflammatory.In some embodiments, the subject is not receiving concurrently for thetreatment or prevention of said uveitis a pharmaceutical compositioncomprising a steroid. In some embodiments, the subject is not receivingconcurrently for the treatment or prevention of said uveitis apharmaceutical composition comprising a non steroid immunosuppressantand a pharmaceutical composition comprising a non-steroidanti-inflammatory. In some embodiments, the subject is not receivingconcurrently for the treatment or prevention of said uveitis apharmaceutical composition comprising a non steroid immunosuppressantand a pharmaceutical composition comprising a steroid. In someembodiments, the subject is not receiving concurrently for the treatmentor prevention of said uveitis a pharmaceutical composition comprising anon-steroid anti-inflammatory and a pharmaceutical compositioncomprising a steroid. In some embodiments, the subject is not receivingconcurrently for the treatment or prevention of said uveitis any of apharmaceutical composition comprising a non steroid immunosuppressant, apharmaceutical composition comprising a non-steroid anti-inflammatoryand a pharmaceutical composition: comprising a steroid. In someembodiments, the non-steroid immunosuppressant is a DNA synthesisinhibitor, a cyclosporine, a mycophenolate or a colchicine. In someembodiments, the DNA synthesis inhibitor is azathioprine, an alkylatingagent, an anti-metabolite (e.g., methotrexate), X-ray therapy,chlorambucil or cyclophosphamide. In some embodiments, the non-steroidanti-inflammatory is a TNF inhibitor, an IL-6 inhibitor or an IL-17inhibitor. In some embodiments, the steroid is a steroid hormoneselected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid.

In some embodiments of each or any of the aforementioned methods, thetreatment refractory uveitis is uveitis that is refractory to treatmentwith a pharmaceutical composition selected from the group consisting ofa pharmaceutical composition comprising a non-steroid immunosuppressant,a pharmaceutical composition comprising a non-steroid anti-inflammatoryand a pharmaceutical composition comprising a steroid. In someembodiments, the treatment refractory uveitis is uveitis that isrefractory to treatment with a pharmaceutical composition comprising anon-steroid immunosuppressant. In some embodiments, the non-steroidimmunosuppressant is a DNA synthesis inhibitor, a cyclosporine, amycophenolate or a colchicine. In some embodiments, the DNA synthesisinhibitor is azathioprine, an alkylating agent, an anti-metabolite(e.g., methotrexate), X-ray therapy, chlorambucil or cyclophosphamide.In some embodiments, the treatment refractory uveitis is uveitis that isrefractory to treatment with a pharmaceutical composition comprising anon-steroid anti-inflammatory. In some embodiment, the non-steroidanti-inflammatory is a TNF inhibitors an IL-6 inhibitor or an IL-17inhibitor. In some embodiments, the treatment refractory uveitis isuveitis that is refractory to treatment with a pharmaceuticalcomposition comprising a steroid. In some embodiments, the steroid is asteroid hormone selected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid.

The present disclosure also provides a method of Inhibiting an acuteuveitis exacerbation (e.g., uveitis flare) in a subject, the methodcomprising administering to the subject an effective amount ofanti-IL-1β antibody or binding fragment thereof, wherein the subject hasreceived prior treatment for uveitis with one or more pharmaceuticalcompositions comprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid. In some embodiments, the non-steroidimmunosuppressant is a DNA synthesis inhibitor, a cyclosporine, amycophenolate or a colchicine. In some embodiments, the DNA synthesisinhibitor is azathioprine, an alkylating agent, an anti-metabolite(e.g., methotrexate), X-ray therapy, chlorambucil or cyclophosphamide.In some embodiments, the non-steroid anti-inflammatory is a TNFinhibitor, an IL-6 inhibitor or an IL-17 inhibitor. In some embodiments,the steroid is a steroid hormone selected from the group consistingprednisone (e.g., methylprenisolone, prednisolone), cortisol,andrenocorticotrophic hormone and a glucocorticoid. In some embodiments,the subject had an adverse reaction or hypersensitivity to said priortreatment of uveitis with one or more pharmaceutical compositionscomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid. In some embodiments, the subject failedsaid prior treatment of uveitis with one or more pharmaceuticalcompositions comprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid. In some embodiments, the subjectpartially responded to said prior treatment of uveitis with one or morepharmaceutical compositions comprising a non-steroid immunosuppressant,a non-steroid anti-inflammatory or a steroid. In some embodiments, theacute uveitis exacerbation has a severity grade of at least a 2 stepincrease in intraocular inflammation according to SUN criteria.

The disclosure also provides a method of inhibiting an acute uveitisexacerbation (e.g., uveitis flare) in a subject, the method comprisingadministering to the subject an effective amount of anti-IL-1β antibodyor binding fragment thereof wherein the subject is receiving concurrenttreatment for said uveitis with at least one (e.g., one or two)pharmaceutical compositions comprising a non-steroid immunosuppressant,a non-steroid anti-inflammatory or a steroid. In some embodiments, thenon-steroid immunosuppressant is a DNA synthesis inhibitor, acyclosporine, a mycophenolate or a colchicine. In some embodiments, theDNA synthesis inhibitor is azathioprine, an alkylating agent, ananti-metabolite (e.g., methotrexate), X-ray therapy, chlorambucil orcyclophosphamide. In some embodiments, the non-steroid anti-inflammatoryis a TNF inhibitor, an IL-6 inhibitor or an IL-17 inhibitor. In someembodiments, the steroid is a steroid hormone selected from the groupconsisting of prednisone (e.g., methylprenisolone, prednisolone),cortisol, andrenocorticotrophic hormone and a glucocorticoid. In someembodiments, the acute uveitis exacerbation has a severity grade of atleast a 2 step increase in intraocular inflammation according to SUNcriteria.

The disclosure also provides a method of inhibiting an acute uveitisexacerbation, in a subject, the method comprising administering to thesubject an effective amount of anti-IL-1β antibody or binding fragmentthereof, wherein the subject Is receiving concurrently for the treatmentor prevention of said uveitis a pharmaceutical composition comprising aninterferon (e.g., IFN-α)

In some embodiments of each or any of the aforementioned methods, saidinhibiting an acute uveitis exacerbation is an increase in the intervalbetween acute uveitis exacerbations (e.g., between two or more acuteuveitis exacerbations). In some embodiments, said inhibiting an acuteuveitis exacerbation is a decrease In the frequency of acute uveitisexacerbations. In some embodiments, said inhibiting an acute uveitisexacerbation is a decrease in the likelihood of experiencing an acuteuveitis exacerbation. In some embodiments, said inhibiting an acuteuveitis exacerbation is preventing an acute uveitis exacerbation. Insome embodiments, said inhibiting an acute uveitis exacerbation istreating an acute uveitis exacerbation. In some embodiments, saidinhibiting an acute uveitis exacerbation is decreasing the severity ofan acute uveitis exacerbation.

The disclosure also provides a method of treating or preventing uveitisin a subject, the method comprising administering to the subject aneffective amount of anti-IL-1β antibody or binding fragment thereof,wherein the subject is receiving concurrently for the treatment orprevention of said uveitis at least one pharmaceutical compositioncomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid, and wherein said method provides areduction (e.g., tapering) in the dosage of said at least onepharmaceutical composition. In some embodiments, the reduction in dosageis a reduction in the dose of said at least one pharmaceuticalcomposition, as compared to the dose prior to administering theanti-IL-1β antibody or binding fragment thereof. In some embodiments,the reduction in dosage is a reduction in the frequency of doses of saidat least one pharmaceutical composition, as compared to the frequency ofdoses prior to administering the anti-IL-1β antibody or binding fragmentthereof. In some embodiments, the reduction in dosage is a redaction incumulative exposure to said at least one pharmaceutical composition overa period of time (e.g., days, weeks, months) after administering theanti-IL-1β antibody or binding fragment thereof, as compared to thecumulative exposure over a similar period of time prior to administeringthe anti-IL-1β antibody or binding fragment thereof. In someembodiments, reduction in cumulative exposure is a reduction in areaunder the curve (e.g., AUG). In some embodiments, the reduction in areaunder the curve is shown by reduced average blood concentration of theat least one pharmaceutical composition over a time-adjusted integratedaverage (e.g., for a time vs. drug dose). In some embodiments, thedosage of a pharmaceutical composition comprising a steroid is reduced.In some embodiments, the dosage of a pharmaceutical compositioncomprising a non-steroid immunosuppressant is reduced. In someembodiments, the dosage of a pharmaceutical composition comprising anon-steroid anti-inflammatory is reduced. In some embodiments, thedosage of at least two pharmaceutical compositions comprising a steroid,non-steroid immunosuppressant or a non-steroid anti-inflammatory isreduced. In some embodiments, the non-steroid immunosuppressant is a DNAsynthesis inhibitor, a cyclosporine, a mycophenolate or a colchicine. Insome embodiments, the DNA synthesis inhibitor is azathioprine, analkylating agent, an anti-metabolite (e.g., methotrexate), X-raytherapy, chlorambucil or cyclophosphamide. In some embodiments, thenon-steroid anti-inflammatory is a TNF inhibitor, an inhibitor or anIL-17 inhibitor. In some embodiments, the steroid is a steroid hormoneselected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid (e.g., dexamethasone).

The disclosure also provides a method of inhibiting an acute uveitisexacerbation (e.g., uveitis flare) in a subject diagnosed with uveitis,the method comprising administering to the subject an effective amountof anti-IL-1β antibody or binding fragment thereof, wherein the uveitisis treatment refractory (e.g., treatment resistant) uveitis and whereinthe acute uveitis exacerbation has a severity grade of at least a 2 stepincrease in intraocular inflammation according to the SUN criteria.

The disclosure also provides a method of inhibiting an acute uveitisexacerbation (e.g., uveitis flare) in a subject diagnosed with uveitis,the method comprising administering to the subject an effective amountof anti-IL-1β antibody or binding fragment thereof, wherein the uveitisis treatment refractory (e.g., treatment, resistant) uveitis and whereinthe acute uveitis exacerbation has a new area of retinitis.

In some embodiments of each or any of the aforementioned methods, thesubject is a subject at risk for an acute uveitis exacerbation.

The disclosure also provides a method of inhibiting (e.g., treating,preventing) uveitis in a subject, the method comprising administering tothe subject an anti-IL-1β antibody or binding fragment thereof in aclose amount and frequency sufficient to maintain a systemic troughserum concentration of at least about 0.5 ug/mL, at least about 1.0ug/mL, at least about 1.5 ug/mL, at least about 2.0 ug/mL, at leastabout 3.0 ug/mL, at least about 4.0 ug/mL or at least about 3.0 ug/mL ofanti-IL-1β antibody or binding fragment thereof. In some embodiments,the anti-IL-1β antibody or binding fragment thereof is administered in adose amount and frequency sufficient to maintain a systemic trough serumconcentration between about 0.5 ug/mL and about 5 ug/mL, between about 1ug/mL and 5 ug/mL, or between about 2 ug/mL and 5 ug/mL.

The disclosure also provides a method of treating uveitis in a subject,the method comprising; 1) diagnosing uveitis in the subject, and 2)administering to the subject of step 1) an effective amount ofanti-IL-1β antibody or binding fragment thereof, wherein said methodresults in an improvement in anterior uveitis or posterior uveitis. Insome embodiments, the method results in an improvement in both anterioruveitis and posterior uveitis. In some embodiments, the subjectdiagnosed with uveitis is a subject diagnosed with panuveitis. In someembodiments, the method further results in an improvement inintermediate uveitis.

The disclosure also provides a method of treating uveitis in a subject,the method comprising: 1) diagnosing uveitis in the subject, and 2)administering to the subject of step 1) an effective amount ofanti-IL-1β antibody or binding fragment thereof wherein said methodresults in an improvement in at least one or two parameters (e.g., atleast three parameters, at least four parameters, at least fiveparameters) selected from visual acuity, vitreous haze, anterior chambercell score, macular edema, laser flare cell count (e.g., flare score),subretinal pooling, epiretinal membrane formation, hypopyon, subretinalneovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, vascular leakage (e.g., fundusfluorescein angiography leakage score, dual fluorescein angiography andindocyanine green angiography score), optic disc hyperfluorescence, discmargin staining, optic disc leakage, cystic pooling, posterior polearcades, retinal capillary nonperfusion, macular ischemia, pinpointleaks, retinal staining, iritis, iridocyclitis, anterior cyclitis, parsplanitis, posterior cyclitis, focal choroiditis, multifocal choroiditis,diffuse choroiditis, chorioretinitis, retinochoroiditis, retinitis,neuroretinitis, retinal dysfunction and elevated intraocular pressure.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous haze, anteriorchamber cell score, macular edema, laser flare cell count (e.g., flarescore), subretinal pooling, epiretinal membrane formation, hypopyon,subretinal neovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, fundus fluorescein angiography leakagescore, optic disc hyperfluorescence, disc margin staining, optic discleakage, cystic pooling, posterior pole arcades, retinal capillarynonperfusion, macular ischemia, pinpoint leaks, retinal staining,iritis, iridocyclitis, anterior cyclitis, pars planitis, posterioreyelids, focal choroiditis, multifocal choroiditis, diffuse choroiditis,chorioretinitis, retinochoroiditis, retinitis and neuroretinitis. Insome embodiments, the method results in an improvement in Ben Ezrascore.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous haze, anteriorchamber cell score, macular edema, laser flare cell count (e.g., flarescore), subretinal pooling, epiretinal membrane formation, hypopyon,subretinal neovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, fundus fluorescein angiography leakagescore, optic disc hyperfluorescence, disc margin staining, optic discleakage, cystic pooling, posterior pole arcades, retinal capillarynonperfusion, macular ischemia, pinpoint leaks and retinal staining. Insome embodiments, the method results in an improvement in Ben Ezrascore.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters atleast three parameters, at least four parameters, at least fiveparameters) selected from visual acuity, vitreous haze, laser flare cellcount (e.g., flare score), retinal infiltrates, retinal vasculitis andoptic disk hyperfluorescence. In some embodiments, the method results inan improvement in Ben Ezra score.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters) selectedfrom visual acuity, vitreous haze, laser flare cell count (e.g., flarescore) and retinal vasculitis. In some embodiments, the method resultsin an improvement in at least two parameters selected from visualacuity, vitreous haze, laser flare cell count (e.g., flare score) andretinal vasculitis. For example, an improvement in one parameter may bean improvement in visual acuity, vitreous haze, laser flare cell count(e.g., flare score) or retinal vasculitis. For example, an improvementin two parameters may be an improvement in two of visual acuity,vitreous base, laser flare cell count (e.g., flare score) or retinal,vasculitis. In some embodiments of each or any of the aforementionedmethods, the method results in an improvement in Ben Ezra score.

The disclosure also provides a method of treating uveitis in a subject,the method comprising: 1) diagnosing uveitis in the subject, and 2)administering to the subject of step 1) an effective amount ofanti-IL-1β antibody or binding fragment thereof, wherein said methodresults in an improvement in at least one of visual acuity, vitreousbase, laser flare cell count (e.g., flare score) and retinal vasculitis.

In some embodiments of each or any of the aforementioned methods, theuveitis in non-infectious uveitis.

In some embodiments of each or any of the aforementioned methods, thesubject has been diagnosed with a disease or condition selected fromBehçet's disease, spondyloarthritides (e.g., ankylosing spondylitis,reactive arthritis), psoriatic arthritis, psoriasis, inflammatory boweldisease, ulcerative colitis, sarcoidosis, tubulointerstitial nephritisand uveitis (TINU) syndrome, rheumatoid arthritis, Kawasaki disease,Sjögren's syndrome, systemic lupus erythematosus, polyarteritis, Reiterdisease, Wegener's granulomatosis, Vogt-Koyanagi-Harada syndrome,systemic juvenile idiopathic arthritis and granulomatous angiitis.

In some embodiments of each or any of the aforementioned methods, thesubject has been diagnosed with cytomegalovirus infection,toxoplasmosis, syphilis, tuberculosis, cat scratch disease, Lymedisease, West Nile virus infection, herpes simplex virus infection,human immunodeficiency virus infection, fungal infection orvaricella-zoster infection.

In some embodiments of each or any of the aforementioned methods, thesubject has been diagnosed with a disease or condition selected frompars planitis, multiple sclerosis, sympathetic ophthalmia, birdshotchoroidopathy, immune recovery uveitis (e.g., immune reconstitutioninflammatory syndrome), lymphoma and idiopathic uveitis.

The present disclosure also provides a method of treating Behçet'sdisease in a subject, the method comprising administering to the subjectan effective amount of anti-IL-1β antibody or binding fragment thereof,wherein the subject has been diagnosed with uveitis and said uveitis istreatment refractory (e.g., treatment resistant) uveitis. In someembodiments, the treatment refractory uveitis is uveitis that isrefractory to treatment with a pharmaceutical composition comprising anon-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid. In some embodiments, the non-steroid immunosuppressant is a DNAsynthesis inhibitor, a cyclosporine, a mycophenolate or a colchicine. Insome embodiments, the DNA synthesis inhibitor is azathioprine, analkylating agent, an anti-metabolite (e.g., methotrexate). X-raytherapy, chlorambucil, or cyclophosphamide. In some embodiments, thenon-steroid anti-inflammatory is a TNF inhibitor, an IL-6 inhibitor oran IL-17 inhibitor. In some embodiments, the steroid is a steroidhormone selected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid.

The disclosure also provides a method of treating Behçet's disease in asubject the method comprising administering to the subject an effectiveamount of anti-IL-1β antibody or binding fragment thereof, said methodfurther comprising treating an acute uveitis exacerbation (e.g., uveitisflare), wherein the subject has been diagnosed with uveitis and saiduveitis is treatment refractory uveitis, and wherein the subject isreceiving concurrent treatment for said acute uveitis exacerbation withone or two pharmaceutical compositions comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid. In someembodiments, the non-steroid immunosuppressant is a DNA synthesisinhibitor, a cyclosporine, a mycophenolate or a colchicine. In someembodiments, the DNA synthesis inhibitor is azathioprine, an alkylatingagent, an anti-metabolite (e.g., methotrexate), X-ray therapy,chlorambucil or cyclophosphamide. In some embodiments, the non-steroidanti-inflammatory is a TNF inhibitor, an IL-6 inhibitor or an IL-17inhibitor. In some embodiments, the steroid is a steroid hormoneselected from the group consisting of prednisone (e.g.,methylprenisolone, prednisolone), cortisol, andrenocorticotrophichormone and a glucocorticoid. In some embodiments, the acute uveitisexacerbation has a severity grade of at least a 2 step increase inintraocular inflammation according to SUN criteria.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in anterior uveitis or posterioruveitis. In some embodiments, the method results in an improvement inboth anterior uveitis and posterior uveitis.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous haze, anteriorchamber cell score, macular edema, laser flare cell count (e.g., flarescore), subretinal pooling, epiretinal membrane formation, hypopyon,subretinal neovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, vascular leakage (e.g., fundusfluorescein angiography leakage score, dual fluorescein angiography andindocyanine green angiography score), optic disc hyperfluorescence, discmargin staining, optic disc leakage, cystic pooling, posterior polearcades, retinal capillary nonperfusion, macular ischemia, pinpointleaks, retinal staining, iritis, iridocyclitis, anterior eyelids, parsplanitis, posterior cyclitis, focal choroiditis, multifocal choroiditis,diffuse choroiditis, chorioretinitis, retinochoroiditis, retinitis,neuroretinitis, retinal dysfunction and elevated intraocular pressure.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous base, anteriorchamber cell score, macular edema, laser flare cell count (e.g., flarescore), subretinal pooling, epiretinal membrane formation, hypopyon,subretinal neovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, fundus fluorescein angiography leakagescore, optic disc hyperfluorescence, disc margin staining, optic discleakage, cystic pooling, posterior pole arcades, retinal capillarynonperfusion, macular ischemia, pinpoint leaks, retinal staining,iritis, iridocyclitis, anterior cyclitis, pars planitis, posteriorcyclitis, focal choroiditis, multifocal choroiditis, diffusechoroiditis, chorioretinitis, retinochoroiditis, retinitis andneuroretinitis.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous haze, anteriorchamber cell score, macular edema, laser flare cell count (e.g., flarescore), subretinal pooling, epiretinal membrane formation, hypopyon,subretinal neovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, fundus fluorescein angiography leakagescore, optic disc hyperfluorescence, disc margin staining, optic discleakage, cystic pooling, posterior pole arcades, retinal capillarynonperfusion, macular ischemia, pinpoint leaks and retinal staining.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters, at leastfive parameters) selected from visual acuity, vitreous haze, laser flarecell count (e.g., flare score), retinal infiltrates, retinal vasculitisand optic disk hyperfluorescence.

In some embodiments of each or any of the aforementioned methods, themethod results in an improvement in at least one or two parameters(e.g., at least three parameters, at least four parameters) selectedfrom visual acuity, vitreous haze, laser flare cell count (e.g., flarescore) and retinal vasculitis. In some embodiments, the method resultsin an improvement in at least two parameters selected from visualacuity, vitreous haze, laser flare cell count (e.g., flare score) andretinal vasculitis. For example, an improvement in one parameter may bean improvement in visual acuity, vitreous haze, laser flare cell count(e.g., flare score) or retinal vasculitis, for example, an improvementin two parameters may be an improvement in two of visual acuity,vitreous haze, laser flare cell count (e.g., flare score) or retinalvasculitis.

In some embodiments each or any of the aforementioned methods, themethod results in an improvement in Ben Ezra score.

In some embodiments of each or any of the aforementioned methods, theantibody or antibody fragment binds to human IL-1β with a dissociationconstant of about 1 nM or less. In some embodiments, the antibody orantibody fragment binds to human IL-1β with a dissociation constant ofabout 250 pM or less. In some embodiments, the antibody or antibodyfragment binds to human IL-1β with a dissociation constant of about 50pM or less. in some embodiments, the antibody or antibody fragment bindsto human IL-1β with a dissociation constant of about 10 pM or less. Insome embodiments, the antibody or antibody fragment binds to human IL-1βwith a dissociation constant of about 1 pM or less. In some embodiments,the antibody or antibody fragment binds to human IL-1β with adissociation constant of about 0.3 pM or less.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof is a neutralizingantibody.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof binds to an IL-1βepitope such that the bound antibody or fragment substantially permitsthe binding of IL-1β to IL-1 receptor I (IL-1R1).

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof does not detectably bindto IL-1α, IL-1R or IL-1Ra.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment, thereof competes with thebinding of an antibody having the light chain, variable region of SEQ IDNO:5 and the heavy chain variable region of SEQ ID NO:6.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof binds to an epitope ofIL-1β that is substantially the same as the epitope bound by an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof binds to an epitopeincorporating Glu64 of IL-1β.

In some embodiments of each or any of the aforementioned methods, theantibody or antibody fragment binds to amino acids 1-34 of the Nterminus of IL-1β.

In some embodiments, each or any of the anti-IL-1β antibody or bindingfragment thereof is Human Engineered or humanized.

In some embodiments, each or any of the anti-IL-1β antibody or bindingfragment thereof is human.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof is administered in oneor more doses of about 3 mg/kg or less of antibody or fragment. In someembodiments, the antibody or antibody fragment is administered in one ormore doses of about 1 mg/kg or less of antibody or fragment. In someembodiments, the antibody or antibody fragment is administered in one ormore doses of about 0.3 mg/kg or less of antibody or fragment. In someembodiments, the antibody or antibody fragment is administered in one ormore doses of about 0.1 mg/kg or less of antibody or fragment. In someembodiments, the antibody or antibody fragment is administered in one ormore doses of about 0.03 mg/kg or less of antibody or fragment. In someembodiments, the antibody or antibody fragment is administered in one ormore doses of about 0.01 mg/kg or less of antibody or fragment. In someembodiments, the one or more doses are at least about 0.01 mg/kg ofantibody or fragment.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof is administered as afixed dose, independent of a dose per subject weight ratio. In someembodiments, the antibody or fragment is administered in one or moredoses of 500 mg or less of antibody or fragment. In some embodiments,the antibody or fragment is administered in one or more doses of 250 mgor less of antibody or fragment. In some embodiments, the antibody orfragment is administered in one or more doses of 100 mg or less ofantibody or fragment. In some embodiments, the antibody or fragment isadministered in one or more doses of 50 mg or less of antibody orfragment. In some embodiments, the antibody or fragment is administeredin one or more doses of 25 mg or less of antibody or fragment, in someembodiments, the antibody or fragment is administered in one or moredoses of 10 mg or less of antibody or fragment. In some embodiments, theantibody or fragment is administered in one or more doses of 1.0 mg orless of antibody or fragment. In some embodiments, the antibody orfragment is administered in one or more doses of at least 1.0 mg ofantibody or fragment. In some embodiments, the antibody or fragment isadministered in one or more doses of at least 10 mg of antibody orfragment. In some embodiments, the antibody or fragment is administeredin one or more doses of about 5 mg to about 150 mg of antibody orfragment, in some embodiments, the antibody or fragment is administeredin one or more doses of about 10 mg to about 75 mg of antibody orfragment (e.g., 20 mg, 30 mg, 40 mg, 50 mg, 60 mg or 70 mg). In someembodiments, the antibody or fragment is administered in one or moredoses of about 20 mg to about 50 mg of antibody or fragment. In someembodiments, the antibody or fragment is administered in one or moredoses of about 30 mg of antibody or fragment.

In some embodiments of each or any of the aforementioned methods,administration of an initial dose of the antibody or antibody fragmentis followed by the administration of one or more subsequent doses. Insome embodiments, administration of an initial dose of the antibody orantibody fragment is followed by the administration of one or moresubsequent closes, and wherein said one or more subsequent doses are inan amount that is approximately the same or less than the initial dose.In some embodiments, administration of an initial dose of the antibodyor antibody fragment is followed by the administration of one or moresubsequent doses, and wherein said one or more subsequent doses are inan amount that is approximately 10% less than an initial dose, 20% lessthan the initial dose, 30% less than the initial dose, 40% less than theInitial dose. 50% less than the initial dose, 60% less than the initialdose, 70% less than the initial dose, 80% less than the initial dose, or90% less than the initial dose, for example, when an Initial dose of 40mg is given, one or more subsequent doses may be 20% less (32 mg), 30%less (28 mg), 40% less (24 mg), 50% less (20 mg), 60% less (16 mg), etc.As another example, when an initial dose of 50 mg is given, one or moresubsequent doses may be 20% less (40 mg), 30% less (35 mg), 40% less (30mg), 50% less (25 mg), 60% (20 mg), etc. As yet another example, when anInitial dose of 60 mg is given, one or more subsequent doses may be 20%less (48 mg), 30% less (42 mg), 40% less (36 mg), 50% less (30 mg), 60%(24 mg), etc. In some embodiments, administration of an initial dose ofthe antibody or antibody fragment is followed by the administration ofone or more subsequent doses, and wherein at least one of the subsequentdoses is in an amount that is more than the initial dose. In someembodiments, administration of an initial dose of the antibody orantibody fragment is followed by the administration of one or moresubsequent doses, and wherein at least one of the subsequent doses is inan amount that is at least 10% more, 20% more, 30% more, 40% more 50%more, 75% more or 100% more than the initial dose. For example, when aninitial dose of 20 mg is given, one or more subsequent doses may be 20%more (24 mg), 30% more (26 mg), 40% more (28 mg), 50% more (30 mg), 100%more (40 mg), etc. As another example, when an initial dose of 30 mg isgiven, one or more subsequent doses may be 20% more (36 mg), 30% more(39 mg), 40% more (42 mg), 50% more (45 mg), 100% more (60 mg), etc. Asyet another example, when an initial dose of 40 mg is given, one or moresubsequent doses may be 20% more (48 mg), 30% more (52 mg), 40% more (56mg), 50% more (60 mg), 100% more (80 mg), etc.

In some embodiments of each or any of the aforementioned methods, theanti-IL-1β antibody or binding fragment thereof is administered in adose amount and frequency sufficient to maintain a systemic trough serumconcentration of at least about 0.5 ug/mL, at least about 1.0 ug/mL, atleast about 1.5 ug/mL, at least about 2.0 ug/mL, at least about 3.0ug/mL, at least about 4.0 ug/mL or at least about 5.0 ug/mL ofanti-IL-1β antibody or binding fragment thereof. In some embodiments,the anti-IL-1β antibody or binding fragment thereof is administered in adose amount and frequency sufficient to maintain a systemic trough serumconcentration between about 0.5 ug/mL and about 5 ug/mL, between about 1ug/mL and 5 ug/mL, or between about 2 ug/mL and 5 ug/mL. In someembodiments of each or any of the aforementioned methods, the anti-IL-1βantibody or binding fragment thereof has a lower IC₅₀ than an IL-1βreceptor antagonist in a human whole blood IL-1β inhibition assay thatmeasures IL-1β induced production of IL-8. In some embodiments, theIL-1β receptor antagonist is anakinra.

The disclosure also provides for use of an anti-IL-1β antibody orbinding fragment thereof which has a lower IC₅₀ than an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8, in the manufacture of a compositionfor use in the treatment of uveitis, wherein the uveitis is treatmentrefractory (e.g., treatment resistant) uveitis. In some embodiments, theIL-1β receptor antagonist is anakinra.

It is to be understood that where the present specification mentionsmethods of treatments making use of antibodies or binding fragmentsthereof with certain properties (such as Kd values or IC₅₀ values), thisalso means to embody the use of such antibodies or fragments thereof inthe manufacture of a medicament for use in these methods. Further, theinvention also encompasses antibodies or binding fragments thereofhaving these properties as well as pharmaceutical compositionscomprising these antibodies or binding fragments thereof for use in themethods of treatment discussed hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing seven subjects from the IL-1β antibodyclinical trial, with information about, treatment medicines receivedprior to enrollment in the study.

FIG. 2 is a table showing clinical data for subject 1001 treated with anIL-1β antibody.

FIG. 3 is a table showing clinical data for subject 1002 treated with anIL-1β antibody,

FIG. 4 is a table showing clinical data for subject 1003 treated with anIL-1β antibody,

FIG. 5 is a table showing clinical data for subject 1004 treated with anIL-1β antibody.

FIG. 6 is a table showing clinical data for subject 1005 treated with anIL-1β antibody.

FIG. 7 is a table showing clinical data for subject 1006 treated with anIL-1β antibody.

FIG. 8 is a table showing clinical data for subject 1007 treated with anIL-1β antibody.

FIG. 9 is images showing resolution of a hypopyon following treatmentwith an IL-1β antibody.

FIG. 10 is Images showing resolution of vitreous haze followingtreatment with an IL-1β antibody.

DETAILED DESCRIPTION

Effective therapies for use in treating or preventing uveitis haveremained an important medical need. The present disclosure providesmethods and materials, and related articles of manufacture, for treatingor preventing uveitis in a subject; including, treatment refractory(e.g., treatment resistant) uveitis, comprising administering to thesubject an effective amount of anti-IL-1β antibody or binding fragmentthereof. Such materials and methods may be used to replace or complementother pharmaceutical approaches as provided herein.

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types.

The interleukin-1 (IL-1) family of cytokines has been implicated inseveral disease states such as rheumatoid arthritis (RA),osteoarthritis, Crohn's disease, ulcerative colitis (UC), septic shock,chronic obstructive, pulmonary disease (COPD), asthma, graft versus hostdisease, atherosclerosis, adult T-cell leukemia, multiple myeloma,multiple sclerosis, stroke, and Alzheimer's disease. IL-1 family membersinclude IL-1α, IL-1β, and IL-1Ra. Although related by their ability tobind to IL-1 receptors (IL-1R1, IL-1R2), each of these cytokines isexpressed by a different gene and has a different primary amino acidsequence. Furthermore, the physiological activities of these cytokinescan he distinguished from each other.

Compounds that disrupt IL-1 receptor signaling have been investigated astherapeutic agents to treat IL-1 mediated diseases, such as for examplesome of the aforementioned diseases. These compounds Include recombinantIL-1Ra (Amgen Inc., Thousand Oaks, Calif.). IL-1 receptor “trap” peptide(Regeneron Inc., Tarrytown, N.Y.), as well as animal-derived IL-1βantibodies and recombinant IL-1β antibodies and fragments thereof.

As noted above, IL-1 receptor antagonist (IL-1Ra) polypeptide has beensuggested for use in the treatment of gout (So et al., 2007, ibid;McGonagle et al., 2007, ibid), but there remains a need for effectivemeans to treat gout, particularly those that do not require daily,repeated injections. An additional challenge for IL-1 receptorantagonist-based therapeutics is the need for such therapeutics tooccupy a large number of receptors, which is a formidable task sincethese receptors are widely expressed on all cells except red blood cells(Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In mostimmune-mediated diseases, such as the diseases disclosed herein, theamount of IL-1β cytokine that is measurable in body fluids or associatedwith activated ceils is relatively low. Thus, a method of treatmentand/or prevention that directly targets the IL-1β ligand is a superiorstrategy, particularly when administering an IL-1β antibody with highaffinity.

The present invention provides methods and related compositions andarticles of manufacture for the treatment and/or prevention, of gout ina subject (e.g., mammalian, human), using an antibody or fragmentthereof specific for IL-1β.

As shown in Example 1 below, we have surprisingly found that such anantibody (e.g., with very high affinity) can be far more potent anInhibitor of the IL-1 pathway than is IL-Ra (e.g., Kineret®), andprovides an opportunity to achieve a therapeutic effect at a lower doseand/or with less frequent -administration than necessary for otherdrugs, such as recombinant IL-1Ra.

Such methods as described herein with an IL-1β antibody or fragment mayinclude the treatment of a subject suffering from gout (e.g., acutegout, chronic gout, refractory gout). The methods also may includepreventing the occurrence of gout (e.g., acute-gout, chronic gout,refractory gout) in an at risk subject.

Antibodies, Humanized Antibodies, and Human Engineered Antibodies

The IL-1 (e.g., IL-1β) binding antibodies of the present disclosure maybe provided as polyclonal antibodies, monoclonal antibodies (mAbs),recombinant antibodies, chimeric antibodies, CDR-grafted antibodies,fully human antibodies, single chain antibodies, and/or bispecificantibodies, as well as fragments, including variants and derivativesthereof, provided by known techniques, including, but not limited toenzymatic cleavage, peptide synthesis or recombinant techniques.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain, and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (λ) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain. The constant region need not be.present in an antigen binding fragment of an antibody, Antigen bindingfragments of an antibody disclosed herein can include Fab, Fab′,F(ab′)₂, and F(v) antibody fragments. As discussed in more detail below,IL-1β binding fragments encompass antibody fragments and antigen-bindingpolypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable region with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable region and the light chainvariable region, respectively, unless otherwise noted. Heavy chain CDRsare referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRsare referred to herein as CDR-L1, CDR-L2, and CDR-L3. Variable regionsand CDRs In an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods forIdentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at www.bioinf.org.uk/abs(maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and VBASE2at www.vbase2.org, as described in Retter et al., Nucl. Acids Res.,33(Database Issue): D671-D674 (2005). The “Kabatman” database web sitealso includes general rules of thumb for identifying CDRs. The term“CDR,” as used herein, is as defined in Kabat et al., Sequences ofImmunological Interest, 5th ed., U.S. Department of Health and HumanServices, 1991, unless otherwise indicated.

Polyclonal, antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet, hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with 1/5 to {fraction1/10)} the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. At 7-14 dayspost-booster injection, the animals are bled and the serum is assayedfor antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

Monoclonal antibody refers to art antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site. In contrast to conventional (polyclonal), antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes). In addition to theirspecificity, the monoclonal antibodies are advantageous in that they aresynthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al., (Nature, 256:495-7, 1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Themonoclonal antibodies may also be isolated from display libraries (e.g.,yeast libraries, phage antibody libraries) using the techniquesdescribed in, for example, Clackson et al., (Nature 352:624-628, 1991),Marks et al., (J. Mol. Biol. 222:581-597, 1991) Hoogenboom (NatBiotechnol. 23:1105-16,2005) and Mondon et al., (Front Biosci.,13:1117-1129, 2008).

In the hybridoma method, a mouse or other appropriate host animal, such,as a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

It is further contemplated that antibodies may be used as smallerantigen binding fragments of the antibody well-known in the art anddescribed herein.

The present disclosure encompasses IL-1 (e.g., IL-1β) binding antibodiesthat include two full length heavy chains and two full length lightchains. Alternatively, the IL-1β binding antibodies can be constructssuch as single chain antibodies or “mini” antibodies that retain bindingactivity to IL-1β. Such constructs can be prepared by methods known inthe art such as, for example, the PCR mediated cloning and assembly ofsingle chain antibodies for expression in E. coli (as described inAntibody Engineering, The practical approach series, J. McCafferty, H.R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press,1996). In this type of construct, the variable portions of the heavy andlight chains of an antibody molecule are PCR amplified from cDNA. Theresulting amplicons are then assembled, for example, in a second PCRstep, through a linker DNA that encodes a flexible protein linkercomposed of the amino acids Gly and Ser. This linker allows the variableheavy and light chain portions to fold in such a way that the antigenbinding pocket is regenerated and antigen is bound with affinities oftencomparable to the parent full-length dimeric immunoglobulin molecule.

The IL-1 (e.g., IL-1β) binding antibodies and binding fragments of thepresent disclosure encompass variants of the exemplary antibodies,fragments and sequences disclosed herein. Variants include peptides andpolypeptides comprising one or more amino acid sequence substitutions,deletions, and/or additions that have the same or substantially the sameaffinity and specificity of epitope binding as one or more of theexemplary antibodies, fragments and sequences disclosed herein. Thus,variants include peptides and polypeptides comprising one or more aminoacid sequence substitutions, deletions, and/or additions to theexemplary antibodies, fragments and sequences disclosed herein wheresuch substitutions, deletions and/or additions do not cause substantialchanges in affinity and specificity of epitope binding. For example, avariant of an antibody or fragment may result from one or more changesto an antibody or fragment, where the changed antibody or fragment hasthe same or substantially the same affinity and specificity of epitopebinding as the starting sequence. Variants may be naturally occurring,such as allelic or splice variants, or may be artificially constructed.Variants may be prepared from the corresponding nucleic acid moleculesencoding said variants. Variants of the present antibodies and IL-1βbinding fragments may have changes in light and/or heavy chain aminoacid sequences that are naturally occurring or are introduced by invitro engineering of native sequences using recombinant DNA techniques.Naturally occurring variants include “somatic” variants which aregenerated in vivo in the corresponding germ line nucleotide sequencesduring the generation of an antibody response to a foreign antigen.

Variants of IL-1 (e.g., IL-1β) binding antibodies and binding fragmentsmay also be prepared by mutagenesis techniques. For example, amino acidchanges may be introduced at random throughout an antibody coding regionand the resulting variants may be screened for binding affinity forIL-1β or for another property. Alternatively, amino acid changes may beintroduced in selected regions of an IL-1β antibody, such as in thelight and/or heavy chain CDRs, and/or in the framework regions, and theresulting antibodies may be screened for binding to IL-1β or some otheractivity. Amino acid changes encompass one or more amino acidsubstitutions in a CDR, ranging from a single amino acid difference tothe introduction of multiple permutations of amino acids within a givenCDR, such as CDR3. In another method, the contribution of each residuewithin a CDR to IL-1β binding may be assessed by substituting at leastone residue within the CDR with alanine, Lewis et al. (1995), Mol.Immunol. 32: 1065-72. Residues which are not optimal for binding toIL-1β may then be changed in order to determine a more optimum sequence.Also encompassed are variants generated by insertion of amino acids toincrease the size of a CDR, such as CDR3. For example, most light chainCDR3 sequences are nine amino acids in length. Light chain sequences inan antibody which are shorter than nine residues may be optimized forbinding to IL-1 β by insertion of appropriate amino acids to increasethe length of the CDR.

Variants may also be prepared by “chain shuffling” of light or heavychains, Marks et al. (1992), Biotechnology 10: 779-83. A single light(or heavy) chain can be combined with a library having a repertoire ofheavy (or light) chains and the resulting population is screened for adesired activity, such as binding to IL-1β. This permits screening of agreater sample of different heavy (or light) chains in combination witha single light (or heavy) chain than is possible with librariescomprising repertoires of both heavy and light chains.

The IL-1 (e.g., IL-1β) binding antibodies and binding fragments of thepresent disclosure encompass derivatives of the exemplary antibodies,fragments and sequences disclosed herein. Derivatives includepolypeptides or peptides, or variants, fragments or derivatives thereof,which have been chemically modified. Examples include covalentattachment of one or more polymers, such as water soluble polymers,N-linked, or O-linked carbohydrates, sugars, phosphates, and/or othersuch molecules. The derivatives are modified in a manner that isdifferent from naturally occurring or starting peptide or polypeptides,either in the type or location of the molecules attached. Derivativesfurther include deletion of one or more chemical groups which arenaturally present on the peptide or polypeptide.

The IL-1β binding antibodies and binding fragments can be bispecific.Bispecific antibodies or fragments can be of several configurations, forexample, bispecific antibodies may resemble single antibodies (orantibody fragments) but have two different antigen binding sites(variable regions). Bispecific antibodies can be produced by chemicaltechniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA, 78: 5807),by “polydoma” techniques (U.S. Pat. No. 4,474,893) or by recombinant DNAtechniques. Bispecific antibodies can have binding specificities for atleast two different epitopes, at least one of which is an epitope ofIL-1β. The IL-1β binding antibodies and binding fragments can also beheteroantibodies. Heteroantibodies are two or more antibodies, orantibody binding fragments (Fab) linked together, each antibody orfragment having a different specificity.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonalantibodies are contemplated for the present IL-1 (e.g., IL-1β) bindingantibodies and binding fragments. DNA is cloned into a bacterialexpression system. One example of such a technique suitable for thepractice of this invention uses a bacteriophage lambda vector systemhaving a leader sequence that causes the expressed Fab protein tomigrate to the periplasmic space (between the bacterial cell membraneand the cell wall) or to be secreted. One can rapidly generate andscreen great numbers of functional Fab fragments for those which bindIL-1β. Such IL-1β binding agents (Fab fragments with specificity for anIL-1β polypeptide) are specifically encompassed within the IL-1β bindingantibodies and binding fragments of the present disclosure.

The present IL-1 (e.g., IL-1β) binding antibodies and binding fragmentscan be humanized or human engineered antibodies. As used herein, ahumanized antibody, or antigen binding fragment thereof, is arecombinant polypeptide that comprises a portion of an antigen bindingsite from a non-human antibody and a portion of the framework and/orconstant regions of a human antibody. A human engineered antibody orantibody fragment is a non-human (e.g., mouse) antibody that has beenengineered by modifying (e.g., deleting, inserting, or substituting)amino acids at specific positions so as to reduce or eliminate anydetectable immunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-graftedantibodies. Chimeric antibodies are antibodies that Include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81;6841-6855 (1984), Boulianne, et al., Nature, 312; 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessImmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human anti-mouse antibodyresponses (MAMA) to the non-human portion of the antibodies. Chimericantibodies can also be produced by splicing the genes from a mouseantibody molecule of appropriate antigen-binding specificity togetherwith genes from a human antibody molecule of appropriate biologicalactivity, such as the ability to activate human complement and mediateADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81: 6851;Neuberger et al. (1984), Nature, 312; 604. One example is thereplacement of a Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody of the Invention can comprise a heavy chain that comprises acontiguous amino acid sequence (e.g., about 5 or more, 10 or more, oreven 15 or more contiguous amino acid residues) from the frameworkregion of a human antibody (e.g., FR-1, FR-2, or FR-3 of a humanantibody) or, optionally, most or all of the entire framework region ofa human antibody. CDR-grafted antibodies and methods for making them aredescribed in, Jones et al., Nature, 321: 522-525 (1986), Riechmann etal. Nature, 332: 323-327 (19S8), and Verhoeyen et al., Science, 239;1534-1536 (1988)). Methods that can he used to produce humanizedantibodies also are described in U.S. Pat. Nos. 4,816,567, 5,721,167,5,837,243, and 6,180,377. CDR-grafted antibodies are considered lesslikely than chimeric antibodies to induce an immune reaction againstnon-human antibody portions. However, it has been reported thatframework sequences from the donor antibodies are required for thebinding affinity and/or specificity of the donor antibody, presumablybecause these framework sequences affect the folding of theantigen-binding portion of the donor antibody. Therefore, when donor,non-human CDR sequences are grafted onto unaltered human frameworksequences, the resulting CDR-grafted antibody can exhibit, in somecases, loss of binding avidity relative to the original non-human donorantibody. See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), andVerhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include for example “veneered” antibodiesand antibodies prepared using HUMAN ENGINEERING™ technology (see forexample, U.S. Pat. Nos. 5,766,886 and 5,869,619), HUMAN ENGINEERING™technology is commercially available, and involves altering an non-humanantibody or antibody fragment, such as a mouse or chimeric antibody orantibody fragment, by making specific changes to the amino acid sequenceof the antibody so as to produce a modified antibody with reducedimmunogenicity in a human that nonetheless retains the desirable bindingproperties of the original non-human antibodies. Generally, thetechnique involves classifying amino acid residues of a non-human (e.g.,mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues.The classification is performed using a global risk/reward calculationthat evaluates the predicted benefits of making particular substitution(e.g., for immunogenicity in humans) against the risk that thesubstitution will affect the resulting antibody's folding and/orantigen-binding properties. Thus, a low risk position is one for which asubstitution is predicted to be beneficial because it is predicted toreduce immunogenicity without significantly affecting antigen bindingproperties. A moderate risk position is one for which a substitution ispredicted to reduce immunogenicity, but is more likely to affect proteinfolding and/or antigen binding. High risk positions contain residuesmost likely to be involved in proper folding or antigen binding.Generally, low risk positions in a non-human antibody are substitutedwith human residues, high risk positions are rarely substituted, andhumanizing substitutions at moderate risk positions are sometimes made,although not indiscriminately, Positions with prolines in the non-humanantibody variable region sequence are usually classified as at leastmoderate risk-positions.

The particular human amino acid residue to be substituted at a given lowor moderate risk position of a non-human (e.g., mouse) antibody sequencecan be selected by aligning an amino acid sequence from the non-humanantibody's variable regions with the corresponding region of a specificor consensus human antibody sequence. The amino acid residues at low ormoderate risk positions in the non-human sequence can be substituted forthe corresponding residues In the human antibody sequence according tothe alignment. Techniques for making human engineered proteins aredescribed in greater detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and5,869,619, and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCOD-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody arid replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (II) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

Exemplary humanized or human engineered antibodies include IgG, IgM,IgE, IgA, and IgD antibodies. The present antibodies can be of any class(IgG, IgA, IgM, IgE, IgD, etc) or isotype and can comprise a kappa orlambda light chain, for example, a human antibody can comprise an IgGheavy chain or defined fragment, such as at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. As a further example, the present antibodies orfragments can comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments can be human antibodies, such asantibodies which bind IL-1β polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,398 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4), immunization of the TC MOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S Patent Publication. No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be generated through the in vitro screening ofantibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol.227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments, that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of IL-1β.

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments—usually Fv or Fabfragments—in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The disclosure contemplates a method for producing target-specificantibody or antigen-binding portion thereof comprising the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with target protein or a portion thereof, isolating phage thatbind target, and obtaining the antibody from the phage. By way ofexample, one method for preparing the library of antibodies for use inphage display techniques comprises the steps of immunizing a non-humananimal comprising human immunoglobulin loci with target antigen or anantigenic portion thereof to create an immune response, extractingantibody producing cells from the immunized animal; isolating RNA fromthe extracted ceils, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using a primer, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant target-specific antibodies of the invention may be obtainedin this way.

Phage-display processes mimic immune selection through the display ofantibody repertoires on the surface of filamentous bacteriophage, andsubsequent selection of phage by their binding to an antigen of choice.One such technique Is described in WO 99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPLand msk receptors using such an approach. Antibodies of the Inventioncan be isolated by screening of a recombinant combinatorial antibodylibrary, preferably a scFv phage display library, prepared using humanV_(L) and V_(H) cDNAs prepared from mRNA derived from human lymphocytes.Methodologies for preparing and screening such libraries are known inthe art. See e.g., U.S. Pat. No. 5,969,108. There are commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Thereare also other methods and reagents that can be used In generating andscreening antibody display libraries (see, e.g., Ladner et al. U.S. Pat.No. 5,223,409; Rang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Maryland et al. PCT Publication No, WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1231;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993)EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;Clackson et al. (1991) Nature 352:024-628; Gram et al. (1992) Proc.Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a Human V_(H) and V_(L)library are screened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 02/01047, McCafferty et al.,(Nature 348:552-554, 1990); and Griffiths et al., (EMBO J 12:725-734,1993). The scFv antibody libraries preferably are screened using targetprotein as the antigen.

Alternatively, the Fd fragment (V_(H)-C_(H)1) and light chain(V_(L)-C_(L)) of antibodies are separately cloned by PCR and recombinedrandomly in combinatorial phage display libraries, which can then beselected for binding to a particular antigen. The Fab fragments areexpressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently.Through several rounds of antigen binding and re-amplification, aprocedure termed panning, Fab specific for the antigen are enriched andfinally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson, T.and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, in vitroselection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193 (2062), and U.S. patent publication no,20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, Ike, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human V_(L) and V_(H) segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedV_(L) and V_(H) segments are screened for target binding, are performedto select preferred V_(L)/V_(H) pair combinations. Additionally, tofurther improve the quality of the antibody, the V_(L) and V_(H)segments of the preferred V_(L)/V_(H) pair(s) can be randomly mutated,preferably within the any of the CDR1, CDR2 or CDR3 region of V_(H)and/or V_(L) in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying V_(L) and V_(H) regions using PCR primerscomplimentary to the V_(H) CDR1, CDR2, and CDR3, or V_(L) CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such, that theresultant PCR products encode V_(L) and V_(H) segments into which randommutations have been introduced into the V_(H) and/or V_(L) CDR3 regions.These randomly mutated V_(L) and V_(H) segments can be rescreened forbinding to target antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the invention, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be earned outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage-display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783, 1992).

Methods for display of peptides on the surface of yeast and microbialcells have also been used to identify antigen specific antibodies. See,for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attachedto yeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingribosome mRNA display methods and microbial ceil display methods.Selection of polypeptide using ribosome display is described in Hanes etal., (Proc. Natl Acad Sci USA, 94:4937-4942, 1997) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is alsouseful for rapid large scale mutational analysis of antibodies. Theselective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

The IL-1 (e.g., IL-1β) binding antibodies and binding fragments maycomprise one or more portions that do not bind IL-1β but instead areresponsible for other functions, such as circulating half-life, directcytotoxic effect, detectable labeling, or activation of the recipient'sendogenous complement cascade or endogenous cellular cytotoxicity. Theantibodies or fragments may comprise all or a portion of the constantregion and may be of any isotype, including IgA (e.g., IgA1 or IgA2),IgD, IgE, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to,or instead of, comprising a constant region, antigen-binding compoundsof the invention may include an epitope tag, a salvage receptor epitope,a label moiety for diagnostic or purification purposes, or a cytotoxicmoiety such as a radionuclide or toxin.

The constant region (when present) of the present antibodies andfragments may be of the γ1, γ2, γ3, γ4, μ, β2, or δ or ε type,preferably of the γ type, more preferably of the γ, type, whereas theconstant part of a human light chain may be of the κ or λ type (whichincludes the λ₁, λ₂ and λ₃ subtypes) but is preferably of the κ type.

Variants also include antibodies or fragments comprising a modified Fcregion, wherein the modified Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. The variant Fc regionmay be designed, relative to a comparable molecule comprising thewild-type Fc region, so as to bind Fc receptors with a greater or lesseraffinity.

For example, the present IL-1β binding antibodies and binding fragmentsmay comprise a modified Fc region, Fc region refers tonaturally-occurring or synthetic polypeptides homologous to the IgGC-terminal domain that is produced upon papain digestion of IgG. IgG Fchas a molecular weight of approximately 50 kD. In the present antibodiesand fragments, an entire Fc region can be used, or only a half-lifeenhancing portion. In addition, many modifications in amino acidsequence are acceptable, as native activity is not in all casesnecessary or desired.

The Fc region can be mutated, if desired, to inhibit its ability to fixcomplement and bind the Fc receptor with high affinity. For murine IgGFc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322renders the protein unable to direct ADCC. Substitution of Glu for Leu235 inhibits the ability of the protein to bind the Fc receptor withhigh affinity. Various mutations for human IgG also are known (see,e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke etal., 1994, The Immunologist 2: 125).

In some embodiments, the present antibodies or fragments are providedwith a modified Fc region where a naturally-occurring Fc region ismodified to increase the half-life of the antibody or fragment in abiological environment, for example, the serum half-life or a half-lifemeasured by an in vitro assay. Methods for altering the original form ofa Fc region of an IgG also are described In U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope Into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope can include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH1, CH3, or V_(H) region, or more than one such region, of theantibody. Alternatively, the epitope is taken from the CH2 domain of theFc region and transferred to the C_(L) region or V_(L) region, or both,of the antibody fragment. See also International applications WO97/34631 and WO 96/32478 which describe Fc variants and theirinteraction with the salvage receptor.

Mutation of residues within Fc receptor binding sites can result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g. replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol Immunol. 30:105-8, 1993).

Antibody fragments are portions of an intact full length antibody, suchas an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies, triabodies,tetrabodies); minibodies; chelating recombinant antibodies; tribodies orbibodies; intrabodies; nanobodies; small modular immunopharmaceuticals(SMIP), adnectins, binding-domain immunoglobulin fusion proteins;camelized antibodies; V_(HH) containing antibodies; and any otherpolypeptides formed from antibody fragments.

The present disclosure includes IL-1β binding antibody fragmentscomprising any of the foregoing heavy or light chain sequences and whichbind IL-1β. The term fragments as used herein refers to any 3 or morecontiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more,or even 10 or more contiguous amino acids) of the antibody andencompasses Fab, Fab′, F(ab′)₂, and F(v) fragments, or the individuallight, or heavy chain variable regions or portion thereof IL-1β bindingfragments include, for example, Fab, Fab′, F(ab′)₂, Fv and scFv. Thesefragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25.These fragments can he produced from intact antibodies using well knownmethods, for example by proteolytic cleavage with enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

In vitro and cell based assays are well described in the art for use indetermining binding of IL-1β to IL-1 receptor type I (IL-1R1), includingassays that determining in the presence of molecules (such asantibodies, antagonists, or other inhibitors) that bind to IL-1β orIL-1R1. (see for example Evans et al., (1995), J. Biol. Chem.270:11477-11483; Vigers et al., (2000), J. Biol. Chem. 275:36927-36933;Yanofsky et al., (1996), Proc. Natl. Acad. Sci. USA 93:7381-7386:Fredericks et al., (2004), Protein Eng. Des. Sel. 17:95-106; Slack etal., (1993), J. Biol. Chem. 268:2513-2524; Smith et al., (2003),Immunity 18:87-96; Vigers et al., (1997), Nature 386:190-194, Ruggieroet al., (1997), J. Immunol. 158:3881-3887; Guo et al., (1995), J. Biol.Chem. 270:27562-27568; Svenson et al., (1995), Eur. J. Immunol.25:2842-2850; Arend et al., (1994), J. Immunol. 153:4766-4774).Recombinant IL-1 receptor type I, including human IL-1 receptor type I,for such assays is readily available from a variety of commercialsources (see for example R&D Systems, SIGMA). IL-1 receptor type I alsocan be expressed from an expression construct or vector introduced intoan appropriate host cell using standard molecular biology andtransfection techniques known in the art. The expressed IL-1 receptortype I may then be isolated and purified for use in binding assays, oralternatively used directly in a cell associated form.

For example, the binding of IL-1β to IL-1 receptor type I may bedetermined by immobilizing an IL-1β binding antibody, contacting IL-1βwith the immobilized antibody and determining whether the IL-1β wasbound to the antibody, and contacting a soluble form of IL-1R1 with thebound IL-1β/antibody complex and determining whether the soluble IL-1R1was bound to the complex. The protocol may also include contacting thesoluble IL-1R1 with the immobilized antibody before the contact withIL-1β, to confirm that the soluble IL-1R1 does not bind to theimmobilized antibody. This protocol can be performed using a Biacore®Instrument for kinetic analysis of binding interactions. Such a protocolcan also be employed to determine whether an antibody or other moleculepermits or blocks the binding of IL-1β to IL-1 receptor type I.

For other IL-1β/IL-1R1 binding assays, the permitting or blocking ofIL-1β binding to IL-1 receptor type I may be determined by comparing thebinding of IL-1β to IL-1R1 in the presence or absence of IL-1βantibodies or IL-1β binding fragments thereof. Blocking is identified inthe assay readout as a designated reduction of IL-1β binding to IL-1receptor type I in the presence of anti-IL-1β antibodies or IL-1βbinding fragments thereof as compared to a control sample that containsthe corresponding buffer or diluent but not an IL-1β antibody or IL-1βbinding fragment thereof. The assay readout may be qualitatively viewedas indicating the presence or absence of blocking, or may bequantitatively viewed as indicating a percent or fold reduction inbinding due to the presence of the antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-1βbinding fragment substantially blocks IL-1β binding to IL-1R1, the IL-1βbinding to IL-1R1 is reduced by at least 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, compared to binding ofthe same concentrations of IL-1β and IL-1R1 in the absence of theantibody or fragment. As another example, when an IL-1β binding antibodyor IL-1β binding fragment substantially permits IL-1β binding to IL-1R1,the IL-1β binding to IL-1R1 is at least about 90%, alternatively atleast about 95%, alternatively at least about 99%, alternatively atleast about 99.9%, alternatively at least about 99.99%, alternatively atleast about 99.999%, alternatively at least about 99.9999%,alternatively substantially identical to binding of the sameconcentrations of IL-1β and IL-1R1 in the absence of the antibody orfragment.

The present disclosure may in certain embodiments encompass IL-1βbinding antibodies or IL-1β binding fragments that bind to the sameepitope or substantially the same epitope as one or more of theexemplary antibodies described herein. Alternatively or additionally,the IL-1β binding antibodies or IL-1β binding fragments compete with thebinding of an antibody having variable region sequences of AB7,described in U.S. application Ser. No. 11/472,813 or WO 2007/002261(sequences shown below). As an example, when an IL-1β binding antibodyor IL-1β binding fragment competes with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region, of SEQ ID NO:6, binding of an antibody having thelight chain variable region, of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6 to IL-1β may be reduced by at least about 2-fold,alternatively at least about 5-fold, alternatively at least about10-fold, alternatively at least about 20-fold, alternatively at leastabout 50-fold, alternatively at least about 100-fold, alternatively atleast about 1000-fold, alternatively at least about 10000-fold, or more,if the binding is measured in the presence of the IL-1β binding antibodyor IL-1β binding fragment. The IL-1β binding antibody or IL-1β bindingfragment may be present in excess of the antibody having the light chainvariable region of SEQ ID NO:5 and the heavy chain variable region ofSEQ ID NO:6, for example an excess of least about 2-fold, alternativelyat least about 5-fold, alternatively at least about 10-foldalternatively at least about 20-fold, alternatively at least about50-fold, alternatively at least about 100-fold, alternatively at leastabout 1000-fold, alternatively at least about 10000-fold. Alternativelyor additionally, the present disclosure encompasses IL-1β bindingantibodies and fragments that bind to an epitope contained in the aminoacid sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1) (U.S. applicationSer. No. 11/472,813, WO 2007/002251) which corresponds to residues83-105 of the mature IL-1β protein. As contemplated herein, one canreadily determine if an IL-1β binding antibody or fragment binds to thesame epitope or substantially the same epitope as one or more of theexemplary antibodies, such as for example the antibody designated AB7,using any of several known methods in the art.

For example, the key amino acid residues (epitope) bound by an IL-1βbinding antibody or fragment may be determined using a peptide array,such as for example, a PepSpot™ peptide array (JPT Peptide Technologies,Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanningthe entire IL-1β amino acid sequence, each peptide overlapping by 11amino acid to the previous one, is synthesized directly on a membrane.The membrane carrying the peptides is then probed with the antibody forwhich epitope binding information is sought, for example at aconcentration of 2 μg/ml, for 2 hr at room temperature. Binding ofantibody to membrane bound peptides may be detected using a secondaryHRP-conjugated goat anti-human (or mouse, when appropriate) antibody,followed by enhanced chemiluminescence (ECL). The peptides spot(s)corresponding to particular amino acid residues or sequences of themature IL-1β protein, and which score positive for antibody binding, areindicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may beperformed and such assays are well known in the art. For example, anantibody of unknown specificity may be compared with any of theexemplary of antibodies (e.g., AB7) of the present disclosure. Bindingcompetition assays may be performed, for example, using a Biacore®instrument for kinetic analysis of binding interactions or by ELISA. Insuch an assay, the antibody of unknown epitope specificity is evaluatedfor its ability to compete for binding against the known comparatorantibody (e.g., AB7). Competition for binding to a particular epitope isdetermined by a reduction in binding to the IL-1β epitope of at leastabout 50%, or at least about 70%, or at least about 80%, or at leastabout 90%, or at least about 95%, or at least about 99% or about 100%for the known comparator antibody (e.g., AB7) and is indicative ofbinding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β bindingregions in exemplary antibodies and/or epitopes recognized by thedisclosed antibodies, it is contemplated that additional antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitycan be generated that parallel the embodiments of this disclosure.

Antigen-binding fragments of an antibody include fragments that retainthe ability to specifically bind to an antigen, generally by retainingthe antigen-binding portion of the antibody. It is well established thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of antigen-bindingportions include (i) a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)² fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment which is theVH and CH1 domains; (iv) a Fv fragment which is the VL and VH domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-540), which is a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso encompassed within the term antigen-binding portion of an antibody.The IL-1β binding antibodies and fragments of the present invention alsoencompass monovalent or multivalent, or monomeric or multimeric (e.g.tetrameric), CDR-derived binding domains with or without a scaffold (forexample, protein or carbohydrate scaffolding).

The present IL-1β binding antibodies or binding fragments may be part oflarger immunoadhesion molecules, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).Antibodies and fragments comprising immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.Preferred antigen binding portions are complete domains or pairs ofcomplete domains.

The IL-1β binding antibodies and binding fragments may also encompassdomain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989)which consist of a V_(H) domain. The IL-1β binding antibodies andfragments of the present invention also encompass diabodies, which arebivalent antibodies in which V_(H) and V_(L) domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see e.g., EP 404,097; WO 93/11161;Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, andPoljak et al., Structure 2:1121-1123, 1994). Diabodies can be bispecificor monospecific.

The IL-1β binding antibodies and binding fragments of the presentdisclosure also encompass single-chain antibody fragments (scFv) thatbind to IL-1β. An scFv comprises an antibody heavy chain variable region(V_(H)) operably linked to an antibody light chain variable region(V_(L)) wherein the heavy chain variable region and the light chainvariable region, together or individually, form a binding site thatbinds IL-1β. An scFv may comprise a V_(H) region at the ammo-terminalend and a V_(L) region at the carboxy-terminal end. Alternatively, scFvmay comprise a V_(L) region at the amino-terminal end and a V_(H) regionat the carboxy-terminal end. Furthermore, although the two domains ofthe Fv fragment, V_(L) and V_(H), are coded for by separate genes, theycan be joined, using recombinant methods, by a synthetic linker thatenables them to be made as a single protein chain in which the V_(L) andV_(H) regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

An scFv may optionally further comprise a polypeptide linker between theheavy chain variable region and the light chain variable region. Suchpolypeptide linkers generally comprise between 1 and 50 amino acids,alternatively between 3 and 12 amino acids, alternatively 2 amino acids.An example of a linker peptide for linking heavy and light chains in anscFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:2). Other examples comprise one or more tandem repeats of this sequence(for example, a polypeptide comprising two to four repeats ofGly-Gly-Gly-Gly-Ser (SEQ ID NO: 2) to create linkers.

The IL-1β binding antibodies and binding fragments of the presentinvention also encompass heavy chain antibodies (HCAb), Exceptions tothe H₂L₂ structure of conventional antibodies occur in some isotypes ofthe immunoglobulins found in camelids (camels, dromedaries and llamas;Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J.Mol. Biol. 275; 413), wobbegong sharks (Nuttall et al., Mol. Immunol.38:313-26, 2001), nurse sharks (Greenberg et al., Nature 374:168-73,1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in thespotted ratfish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; acase of evolutionary innovation,” 2002 Immunogenics 54(1): 39-47). Theseantibodies can apparently form antigen-binding regions using only heavychain variable regions, in that these functional antibodies are dimersof heavy chains only (referred to as “heavy-chain antibodies” or“HCAbs”). Accordingly, some embodiments of the present IL-1β bindingantibodies and fragments may be heavy chain antibodies that specificallybind to IL-1β. For example, heavy chain antibodies that, are a class ofIgG and devoid of light chains are produced by animals of the genusCamelidae which includes camels, dromedaries and llamas(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have amolecular weight of about 95 kDa instead of the about 160 kDa molecularweight of conventional IgG antibodies. Their binding domains consistonly of the heavy-chain variable domains, often referred to as V_(HH) todistinguish them from conventional V_(H). Muyldermans et al., J. Mol.Recognit. 12:131-140 (1999). The variable domain of the heavy-chainantibodies is sometimes referred to as a nanobody (Cortez-Retamozo etal., Cancer Research 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described in Conrath et al.,(Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinantmethods.

Since the first constant domain (C_(H1)) is absent (spliced out duringmRNA processing due to loss of a splice consensus signal), the variabledomain (V_(HH)) is immediately followed by the hinge region, the C_(H2)and the C_(H3) domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999):Woolven et al., Immunogenetics 50:98-101 (1999)). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, Ilama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the Ilama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains.

Although the HCAbs are devoid of light chains, they have anantigen-binding repertoire. The genetic generation mechanism of HCAbs isreviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001) and Nguyen etal. Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark,display similar antigen receptor-containing single monomeric V-domains.Irving el al., J. Immunol. Methods 248:31-45 (2001); Roux el al., Proc.Natl. Acad. Sci. USA 95:11804 (1998).

V_(HH)s comprise small intact antigen-binding fragments (for example,fragments that are about 15 kDa, 118-136 residues). Camelid V_(HH)domains have been found to bind to antigen with high affinity (Desmyteret al., J. Biol. Chem. 276:26283-90, 2001), with V_(HH) affinitiestypically in the nanomolar range and comparable with those of Fab andscFv fragments. V_(HH)s are highly soluble and snore stable than thecorresponding derivatives of scFv and Fab fragments. V_(H) fragmentshave been relatively difficult to produce in soluble form, butimprovements in solubility and specific binding can be obtained whenframework residues are altered to be more V_(HH)-like. (See, forexample, Reichman et al., J Immunol Methods 1999, 231:25-38.) V_(HH)scarry amino acid substitutions that make them more hydrophilic andprevent prolonged interaction with BiP (immunoglobulin heavy-chainbinding protein), which normally hinds to the H-chain in the EndoplasmicReticulum (BR) during folding and assembly, until it is displaced by theL-chain. Because of the V_(HH)s' increased hydrophilicity, secretionfrom the ER is improved.

Functional V_(HH)s may be obtained by proteolytic cleavage of HCAb of animmunized camelid, by direct cloning of V_(HH) genes from B-cells of animmunized camelid resulting in recombinant V_(HH)s, or from naive orsynthetic libraries. V_(HH)s with desired antigen specificity may alsobe obtained through phage display methodology. Using V_(HH)S in phagedisplay is much simpler and more efficient compared to Fabs or scFvs,since only one domain needs to be cloned and expressed to obtain afunctional antigen-binding fragment. Muyldermans, Biotechnol.,74:277-302 (2001): Ghahroudi et al., FEBS Lett. 414:521-520 (1997); andvan der Linden et al., J. Biotechnol. 80:261-270 (2000). Methods forgenerating antibodies having camelid heavy chains are also described inU.S. Patent Publication Nos. 20050136049 and 20050037421.

Ribosome display methods may be used to identify and isolate scFv and/orV_(L)s molecules having the desired binding activity and affinity.Irving et al., J. Immunol. Methods 248:31-45 (2001). Ribosome displayand selection has the potential to generate and display large libraries(10¹⁴).

Other embodiments provide V_(HH)-like molecules generated through theprocess of camelsation, by modifying non-Camelidae V_(H)s, such as humanV_(HH)s, to improve their solubility and prevent non-specific binding.This is achieved by replacing residues on the V_(L)s side of V_(H)s withV_(HH)-like residues, thereby mimicking the more soluble V_(HH)fragments. Camelised V_(H) fragments, particularly those based on thehuman framework, are expected to exhibit a greatly reduced immuneresponse when administered in vivo to a patient and, accordingly, areexpected to have significant advantages for therapeutic applications.Davies et al., FEBS Lett. 339:285-290 (1994); Davies et al., ProteinEng. 9:531-537 (1996); Tanha et al., J. Biol. Chem. 276:24774-24780(2001); and Riechmann et al., Immunol. Methods 231:25-38 (1999).

A wide variety of expression systems are available for the production ofIL-1β binding fragments including Fab fragments, scFv, and V_(HH)s. forexample, expression systems of both prokaryotic and eukaryotic originmay be used for the large-scale production of antibody fragments andantibody fusion proteins. Particularly advantageous are expressionsystems that permit the secretion of large amounts of antibody fragmentsinto the culture medium.

Production of bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For dibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. A “minibody”consisting of scFv fused to CH3 via a peptide linker (hingeless) or viaan IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

The IL-1β binding antibodies and binding fragments also encompassantibodies that are SMIPs or binding domain immunoglobulin fusionproteins specific for target protein. These constructs are single-chainpolypeptides comprising antigen binding domains fused to immunoglobulindomains necessary to carry out antibody effector functions. See e.g.,WO03/041600, U.S. Patent publication 20030133939 and US PatentPublication 20030118592.

IL-1β binding antibodies and binding fragments of the present disclosurealso encompass immunoadhesins. One or more CDRs may be incorporated intoa molecule either covalently or noncovalently to make it animmunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of alarger polypeptide chairs, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRsdisclosed herein permit the immunoadhesin to specifically bind to IL-1β.

The IL-1β binding antibodies and fragments also encompass antibodymimics comprising one or more IL-1β binding portions built on an organicor molecular scaffold (such as a protein or carbohydrate scaffold).Proteins having relatively defined three-dimensional structures,commonly referred to as protein scaffolds, may be used as reagents forthe design of antibody mimics. These scaffolds typically contain one ormore regions which are amenable to specific or random sequencevariation, and such sequence randomization is often carried out toproduce libraries of proteins from which desired products may beselected. For example, an antibody mimic can comprise a chimericnon-immunoglobulin binding polypeptide having an immunoglobulin-likedomain containing scaffold having two or more solvent exposed loopscontaining a different CDR from a parent antibody inserted into each ofthe loops and exhibiting selective binding activity toward a ligandbound by the parent antibody. Non-immunoglobulin protein scaffolds havebeen proposed for obtaining proteins with novel binding properties.(Tramontano et al. J. Mol. Recognit. 7:9, 1994; McConnell and Hoess, J.Mol. Biol. 250:460, 1995). Other proteins have been tested as frameworksand have been used to display randomized residues on alpha helicalsurfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al.,Protein Eng. 8:601, 1995), loops between alpha helices in alpha helixbundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), andloops constrained by disulfide bridges, such as those of the smallprotease inhibitors (Markland et al., Biochemistry 35:8045, 1996;Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene164:243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995). Methods foremploying scaffolds for antibody mimics are disclosed in U.S. Pat. No.5,770,380 and US Patent Publications 2004/0171116, 2004/0266993, and2005/0038229.

Preferred IL-1β antibodies or antibody fragments for use in accordancewith the Invention generally bind to human IL-1β with high affinity(e.g., as determined with BIACORE, as determined by KinExA), such as forexample with an equilibrium binding dissociation constant (K_(D)) forIL-1β of about 10 nM or less, about 5 nM or less, about 2 nM or less, orpreferably about 1 nM or less, about 500 pM or less, or more preferablyabout 250 pM or less, about 100 pM or less, about 50 pM or less, about25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM orless about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, orabout 0.3 pM or less. The dissociation constant may be measured, forexample, using Biacore (GE Healthcare), and measurement using Biacoremay be preferred when the dissociation constant is greater than about 10pM. Alternatively or in addition, the dissociation constant may bemeasured using KinExA (Sapidyne Instruments, Inc), and measurement usingKinExA may be preferred when the dissociation constant is less thanabout 10 pM.

Antibodies or fragments of the present invention may, for example, bindto IL-1β with an IC₅₀ of about 10 nM or less, about 5 nM or less, about2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM orless, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nMor less, as determined by enzyme linked immunosorbent assay (ELISA).Preferably, the antibody or antibody fragment of the present inventiondocs not cross-react with any target other than IL-1. for example, thepresent antibodies and fragments may bind to IL-1β, but do notdetectably bind to IL-1α, or have at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times) greater selectivity in its binding of IL-1β relative to itsbinding of IL-1α. Antibodies or fragments used according to theinvention may, in certain embodiments, inhibit IL-1β induced expressionof serum-IL-6 in an animal by at least 50% (e.g., at least 60%, at least70%, or even at least 80%) as compared to the level of serum IL-6 in anIL-1β stimulated animal that has not been administered an antibody orfragment of the invention. Antibodies may bind IL-1β but permit orsubstantially permit the binding of the bound IL-1β ligand to IL-1receptor type 1 (IL-1R1). In contrast to many known IL-1β binding,antibodies, that block or substantially interfere with binding of IL-1βto IL-1R1, the antibodies designated AB5 and AB7 (U.S. Pat. No.7,531,166) selectively bind to the IL-1β ligand, but permit the bindingof the bound IL-1β ligand to IL-1R1. For example, the antibodydesignated AB7 binds to an IL-1β epitope but still permits the boundIL-1β to bind to IL-1R1. In certain embodiments, the antibody maydecrease the affinity of interaction of bound IL-1β to bind to IL-1R1.Accordingly, the disclosure provides, in a related aspect, use of anIL-1β binding antibody or IL-1β binding antibody fragment that has atleast one of the aforementioned characteristics. Any of the foregoingantibodies, antibody fragments, or polypeptides of the invention can behumanized or human engineered, as described herein.

A variety of IL-1 (e.g., IL-1β) antibodies and fragments known in theart may be used as provided by the disclosure herein, including forexample antibodies and antibody binding fragments (e.g., V-regionsequences) described in, or derived using methods described in thefollowing patents and patent applications: U.S. Pat. No. 4,935,343; US2003/0026806; US 2003/0124617 (e.g., antibody AAL160); U.S. Pat. No.7,566,772 (e.g., antibody 9.5.2); WO 03/034984; WO 95/01997 (e.g.,antibody SK48-E26 VTKY); U.S. Pat. No. 7,446,175 (e.g., antibody ACZ885); WO 03/010282 (e.g., antibody Hu007); WO 03/073982 (e.g., antibodyN55S), U.S. Pat. No. 7,541,033 (e.g., W17, U43, W13, W18, W20), U.S.Pat. No. 7,491,392, WO 2004/072116, WO 2004/067568, EP 0 267 611 B1, EP0 364 778 B1, and U.S. application Ser. No. 11/472,813. As anon-limiting example, antibodies AB5 and AB7 (U.S. Pat. No. 7,531,166)may be used in accordance with the invention. Variable region sequencesof AB5 and AB7 (also referred to as XOMA 052) are as follows:

AB5 LIGHT CHAIN (SEQ ID NO: 3)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCLQGKMLPWTF GGGTKLEIK

The underlined sequences depict (from left to right) CDR1,2 and 3.

HEAVY CHAIN (SEQ ID NO: 4)QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCARNRYDPPWFVDWGQGTLVTVSS 

The underlined sequences depict (from left to right) CDR 1,2 and 3.

AB7 LIGHT CHAIN (SEQ ID NO: 5)DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTF GQGTKLEIK

The underlined sequences depict (from left to right) CDR1, 2 and 3.

HEAVY CHAIN (SEQ ID NO: 6)QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCA RNRYDPPWFVDWGQGTLVTVSS

The underlined sequences depict (from left to right) CDR1, 2 and 3.

The antibodies and antibody fragments described herein can be preparedby arty suitable method. Suitable methods for preparing such antibodiesand antibody fragments are known in the art. Other methods for preparingthe antibodies and antibody fragments are as described herein as part ofthe invention. The antibody, antibody fragment, or polypeptide of theinvention, as described herein, can be isolated or purified to anydegree. As used herein, an isolated compound is a compound that has beenremoved from its natural environment. A purified compound is a compoundthat has been increased in purity, such that the compound exists in aform that Is more pure than it exists (i) in its natural environment or(ii) when initially synthesized and/or amplified under laboratoryconditions, wherein “purity” is a relative term and does not necessarilymean “absolute purity.”

Compositions

IL-1 (e.g., IL-1β) binding antibodies and binding fragments can heformulated in compositions, especially pharmaceutical compositions, foruse in the methods disclosed herein. Such compositions comprise atherapeutically or prophylactically effective amount of art IL-1βbinding antibody or antibody fragment in admixture with a suitablecarrier, e.g., a pharmaceutically acceptable agent. Typically, IL-1βbinding antibodies and binding fragments are sufficiently purified foradministration to an animal (e.g., human) before formulation in apharmaceutical composition.

Pharmaceutically acceptable agents include for example, carriers,excipients, diluents, antioxidants, preservatives, coloring, flavoringand diluting agents, emulsifying agents, suspending agents, solvents,fillers, bulking agents, buffers, delivery vehicles, tonicity agents,cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, and surfactants.

Neutral buffered saline or saline mixed with albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorohexidine, sorbic acid and the like. Hydrogen peroxide also can beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A.R. Gennaro, ed., Mack Publishing Company, 1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 1.0 mM to about 400 mM. In anotherembodiment, a surfactant is included, such as for example, nonionicsurfactants and ionic surfactants such as polysorbates (e.g. polysorbate20, polysorbate 80); poloxamers (e.g. poloxamer 188); poly (ethyleneglycol) phenyl ethers (e.g. Triton); sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-,linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-laurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions can be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, intrarectal, transdermal, oral, andinhaled routes. A parenteral formulation typically will be a sterile,pyrogen-free, isotonic aqueous solution, optionally containingpharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils, intravenous vehicles include fluid and nutrient replenishes,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Pharmaceutical compositions described herein can be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) sustainedrelease and/or increased stability or half-life in a particular localenvironment. The invention contemplates that in certain embodiments suchcompositions may include a significantly larger amount of antibody orfragment in the initial deposit, while the effective amount of antibodyor fragment actually released and available at any point in time for isin accordance with the disclosure herein an amount much lower than theinitial deposit. The compositions can include the formulation of IL-1βbinding antibodies, antibody fragments, nucleic acids, or vectors of theinvention with particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc., as well as agents such as abiodegradable matrix, injectable microspheres, microcapsular particles,microcapsules, bioerodible particles beads, liposomes, and implantabledelivery devices that provide for the controlled or sustained release ofthe active agent which then can be delivered as a depot injection.Techniques for formulating such sustained- or controlled-delivery meansare known and a variety of polymers have been developed and used for thecontrolled release and delivery of drugs. Such polymers are typicallybiodegradable and biocompatible. Polymer hydrogels, including thoseformed by complexation of enantiomeric polymer or polypeptide segments,and hydrogels with temperature or pH sensitive properties, may bedesirable for providing drug depot effect because of the mild andaqueous conditions involved in trapping bioactive protein agents (e.g.,antibodies). See, for example, the description of controlled releaseporous polymeric microparticles for the delivery of pharmaceuticalcompositions in PCT Application Publication WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon- (rhIFN-), interleukin-2, and MN rgp120, Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27; 1221-1223 (1993);Hora et al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 90/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic-acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al. Biopolymers 22, 547 [1983], R. Langer et al.Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18,24 [2000], and Dai et al.Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present invention. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humour of the eye. Esterified derivatives of hyaluronic acidhave been used to produce microspheres for use in delivery that arebiocompatible and biodegrable (see for example, Cortivo et al.,Biomaterials (1991) 12:727-730; European Publication No. 517,565;International Publication No. WO 96/29998; Illum et al., J. ControlledRel. (1994) 29:133-141). Exemplary hyaluronic acid containingcompositions of the present invention comprise a hyaluronic acid esterpolymer in an amount of approximately 0.1% to about 40% (w/w) of anIL-1β binding antibody or fragment to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices can be usedto deliver compositions in accordance with the invention, and suchpolymeric matrices may comprise natural or synthetic polymers.Biodegradable matrices are preferred. The period of time over whichrelease occurs is based on selection of the polymer. Typically, releaseover a period ranging from between a few hours and three to twelvemonths is most desirable. Exemplary synthetic polymers which can be usedto form the biodegradable delivery system include: polymers of lacticacid and glycolic acid, polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid),poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,polyethylene glycol), polyethylene oxide), poly(ethylene terephthalate),poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride,polystyrene and polyvinylpyrrolidone. Exemplary natural polymers includealginate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof (substitutions, additions ofchemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art), albumin and other hydrophilic proteins, zein and otherprolamines and hydrophobic proteins, copolymers and mixtures thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion. The polymeroptionally is in the form of a hydrogel (see for example WO 04/009664,WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587,) thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such, asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324).

A pharmaceutical composition comprising an IL-1β binding antibody orbinding fragment can be formulated for inhalation, such as for example,as a dry powder. Inhalation solutions also can be formulated in aliquefied propellant for aerosol delivery, In yet another formulation,solutions may be nebulized. Additional pharmaceutical composition forpulmonary administration include, those described, for example, in PCTApplication Publication WO 94/20069, which discloses pulmonary deliveryof chemically modified proteins. For pulmonary delivery, the particlesize should be suitable for delivery to the distal lung. For example,the particle size can be from 1 μm to 5 μm; however, larger particlesmay be used, for example, if each particle is fairly porous.

Certain formulations containing IL-1β binding antibodies or antibodyfragments can be administered orally. Formulations administered in thisfashion can be formulated with or without those carriers customarilyused in the compounding of solid dosage forms such as tablets andcapsules. For example, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofa selective binding agent. Diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders also can be employed.

Another preparation can involve an effective quantity of an IL-1βbinding antibody or binding fragment in a mixture with nontoxicexcipients which are suitable for the manufacture of tablets. Bydissolving the tablets in sterile water, or another appropriate vehicle,solutions can be prepared in unit dose form. Suitable excipientsinclude, but are not limited to, inert diluents, such as calciumcarbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate: or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentInvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

Additional formulations will be evident in light of the presentdisclosure, including formulations involving IL-1β binding antibodiesand antibody fragments in combination with one or more other therapeuticagents. For example, in some formulations, an IL-1β binding antibody,antibody fragment (e.g., binding fragment), nucleic acid, or vector ofthe invention is formulated with a second inhibitor of an IL-1 signalingpathway. Representative second inhibitors include, but are not limitedto, antibodies, antibody fragments, peptides, polypeptides, compounds,nucleic acids, vectors sod pharmaceutical compositions, such as, forexample, those described in U.S. Pat. No. 6,899,878, US 2003022869, US20060094663, US 20050186615, US 20030166069, WO/04022718, WO/05084696,WO/05019259. For example, a composition may comprise an IL-1β bindingantibody, antibody fragment, nucleic acid, or vector of the invention incombination with another IL-1β binding antibody, fragment, or a nucleicacid or vector encoding such an antibody or fragment.

The pharmaceutical compositions can comprise IL-1β binding antibodies orbinding fragments thereof in combination with other active agents (e.g.,other than IL-1β binding antibodies or binding fragments).Alternatively, the pharmaceutical compositions can comprise IL-1βbinding antibodies or binding fragments thereof in combination withother pharmaceutical compositions, including, for example,pharmaceutical compositions comprising one or more active agents otherthan IL-1β binding antibodies or binding fragments). Such combinationsare those useful for their intended purpose. The combinations which arepart of this invention can be IL-1β antibodies and fragments, such asfor example those described herein, and at least one additional agent.Examples of active agents that may be used in combination set forthbelow are illustrative for purposes and not intended to be limited. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its Intended function.

The disclosure further contemplates that additional pharmaceuticalcompositions comprising one or more other active agents may beadministered separately from the IL-1β binding antibodies or fragments(e.g. concurrent treatment regimen, subject receiving concurrenttreatment), and such separate administrations may be performed at thesame time or at different times, such as for example the same ordifferent days, or different times of the same day. Administration ofthe other pharmaceutical compositions and/or active agents may beaccording to standard medical practices known in the art, or theadministration may be modified (e.g., longer intervals between doses,smaller dosage levels, delayed initiation) when used in conjunction withadministration of IL-1β binding antibodies or binding fragments, such asdisclosed herein.

Pharmaceutical compositions contemplated in the present disclosureinclude, for example, pharmaceutical compositions comprising one or moreactive agents, including for example, a non-steroid immunosuppressant, anon-steroid anti-inflammatory and/or a steroid. In some embodiments, thenon-steroid immunosuppressant is selected from a nucleic acid (e.g.,DNA) synthesis inhibitor, a cyclosporine, a mycophenolate and acolchicine. In some embodiments, the nucleic acid (e.g., DNA) synthesisinhibitor is azathioprine, an alkylating agent, an anti-metabolite(e.g., methotrexate), X-ray therapy, chlorambucil or cyclophosphamide.In some embodiments, the steroid is a steroid hormone selected fromprednisone (e.g., methylprenisolone, prednisolone), cortisol,andrenocorticotrophic hormone and a glucocorticoid. In some embodiments,the non-steroid anti-inflammatory is a TNF inhibitor, an IL-6 inhibitoror an IL-17 inhibitor.

Other active agents may include, for example, indomethacin,non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin,ibuprofen, and other propionic acid derivatives (alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenelozic acid, fentiazac, fuirofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone).Other combinations include cyclooxygenase-2 (COX-2) inhibitors,aquaretics, oral glucocorticoids, intra-articular glucocorticoids,colchicine, xanthine-oxidase inhibitors, allopurinol, uricosuric agents,sulfinpyrazone, febuxostat, probenecid, fenofibrate, benemid,angiotensin II receptor antagonists, losartan, thiazides, PEG-uricase,sodium bicarbonate, ethylenediaminetetraacetic acid. Other active agentsfor combination include steroids such as prednisolone, prednisone,melhylprednisolone, betamethasone, dexamethasone, or hydrocortisone.Such a combination may be especially advantageous, since one or moreside-effects of the steroid can be reduced or even eliminated bytapering the steroid dose required when treating patients in combinationwith the present antibodies and fragments.

Pharmaceutical compositions (e.g., comprising an active agent) mayinclude, for example antimetabolites, such as for example, methotrexate,azathioprine, mycophenolate mofetil, pyrimidine analogues, purineanalogues, folate antagonists; T-cell inhibitors/calcineurin inhibitors,such as for example, cycloserine, mycophenolate, FK506/Tacrolimus;alkylating/cytotoxic agents, such as for example, cyclophosphamide,chlorambucil; Intravenous Immunoglobulin; biologic agents, such as forexample, Infliximab, Adalimumab, Etanercept; Interleukin-2 receptorantagonists, such as for example, Daclizumab; and Interferon-alpha.

It is further contemplated that an anti-IL-1β antibody or bindingfragment administered to a subject in accordance with the disclosure maybe administered in combination (e.g., concurrently) with treatment withat least one additional pharmaceutical composition (e.g., comprising anactive agent), such as for example any of the aforementioned activeagents. In one embodiment, treatment with the at least one active agentis maintained. In another embodiment, treatment with the at least oneactive agent is reduced (e.g., tapered) or discontinued (e.g., when thesubject is stable) during the course of IL-1β antibody treatment (e.g.,with the anti-IL-1β) antibody or fragment maintained at a constantdosing regimen). In another embodiment, treatment with the at least oneactive agent is reduced (e.g., tapered) or discontinued (e.g., when thesubject is stable), and treatment with the anti-IL-1β antibody orfragment is reduced (e.g., lower dose, less frequent dosing, shortertreatment regimen). In another embodiment, treatment with the at leastone active agent is reduced (e.g., tapered) or discontinued (e.g., whenthe subject is stable), and treatment with the anti-IL-1β antibody orfragment is increased (e.g., higher dose, more frequent dosing, longertreatment regimen). In yet another embodiment, treatment with the atleast one active agent is maintained and treatment with the anti-IL-1βantibody or fragment is reduced or discontinued (e.g., lower dose, lessfrequent dosing, shorter treatment regimen). In yet another embodiment,treatment with the at least one active agent and treatment with theanti-IL-1β antibody or fragment, are reduced or discontinued (e.g.,lower dose, less frequent dosing, shorter treatment regimen).

In some embodiments, reducing the treatment with at least one activeagent (e.g. other than anti-IL-1β antibody or binding fragment) isreduction in the cumulative amount of active agent administered during acourse of treatment. In some embodiments, reducing the treatment with atleast one active agent (e.g., other than anti-IL-1β antibody or bindingfragment) is a reduction in the actual dose amount of active agentadministered. In some embodiments, reducing the treatment with at leastone active agent provides a reduction in systemic immunosuppression.

The pharmaceutical compositions used in the disclosure may include atherapeutically effective amount or a prophylactically effective amountof the IL-1β binding antibodies or binding fragments. A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A prophylactically effectiveamount refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result.

A therapeutically or prophylactically effective amount of apharmaceutical composition comprising an IL-1β binding antibody orfragment will depend, for example, upon the therapeutic objectives suchas the indication for which the composition is being used, the route ofadministration, and the condition of the subject. Pharmaceuticalcompositions are administered in a therapeutically or prophylacticallyeffective amount to treat an IL-1 related condition.

Methods of Use

Anti-IL-1β binding antibodies or binding fragments thereof in atherapeutically effective amount may be used as disclosed herein for thetreatment and/or prevention of uveitis, including, for example,refractory uveitis. The present disclosure also contemplates the use ofother IL-1 pathway inhibitors, as an alternative or in addition to theanti-IL-1β antibodies or fragments.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, as used herein with respect to the methods asdescribed refer to preventing, suppressing, delaying or reducing, eithertemporarily or permanently, either partially or completely, the onset ofa clinical symptoms or manifestation of an event, disease or condition(e.g., in an at risk subject, in a subject with a history of a priorevent, disease or condition), such as, for example, uveitis (e.g., acuteuveitis exacerbation). Such preventing or suppressing need not beabsolute to be useful.

The terms “treatment”, “treat” and “treating” as used with respect tomethods as described herein refer to eliminating, reducing, suppressingor ameliorating, either temporarily or permanently, either partially orcompletely, a clinical symptom, manifestation or progression of anevent, disease or condition (e.g., diagnosed symptom, manifestation orprogression of an event, disease or condition), such as, for example,uveitis (e.g., acute uveitis exacerbation). Such treating need not beabsolute to be useful.

The terms “inhibit”, “inhibiting” and “inhibition” as used herein withrespect to the methods as described refer to preventing, delaying,suppressing, reducing, treating, eliminating or ameliorating, eithertemporarily or permanently, either partially or completely, a clinicalsymptom or manifestation of an event, disease or condition, such, as,for example, uveitis (e.g., acute uveitis exacerbation). Suchpreventing, treating, suppressing or reducing need not be absolute to beuseful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm, of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method or compound of the disclosure.

The term “effective amount” as used herein refers to an amount of acompound (e.g., IL-1β antibody), either alone or as a part of apharmaceutical composition, that is capable of having any detectable,positive effect on any symptom, aspect, parameter or characteristics ofa disease state or condition when administered to a subject (e.g., asone or more doses), including, for example, improving a uveitisparameter as referred to herein. Such effect need not be absolute to bebeneficial.

The terms “treatment refractory” and “treatment resistant” uveitis asused herein refers to chronic or recurrent uveitis (e.g., acute uveitisexacerbation, uveitis flare) in a subject who has received priortreatment for the uveitis with one or more pharmaceutical compositionsincluding, for example, a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid, but not including an IL-1β antibody orbinding fragment. Treatment refractory and treatment resistant uveitisincludes uveitis in a subject, wherein the subject may have bad anadverse reaction or hypersensitivity to the prior treatment, oralternatively or in addition, the subject may have failed or partiallyresponded to the prior treatment (e.g., inadequate or partialtherapeutic effect, inadequate response, insufficient response,incomplete response, partial response).

The terms “reduction in the dosage”, “reduction in dosage”, “dosagereduction” and “reduced dosage” as used herein refers to a change in aprevention or treatment regimen for a pharmaceutical composition, ascompared to a previous prevention or treatment regimen for the samepharmaceutical composition (e.g., prior to administering an anti-IL-1βantibody or binding fragment thereof). Preferably, such change inprevention or treatment regimen is a decrease in some aspect of theprevention or treatment regimen, such as for example, a decrease (e.g.,reduction) in the dose (e.g., amount), a decrease (e.g., reduction) inthe frequency of doses, or a decrease (e.g., reduction) in thecumulative exposure (e.g., area under the curve, AUC) over a period oftime. Such change in prevention or treatment regimen for apharmaceutical composition may be a change in a prevention, or treatmentregimen as compared to a previous prevention or treatment regimen in thesame subject, or alternatively or in addition, a change in a preventionor treatment regimen for a pharmaceutical composition when usedconcurrently with an anti-IL-1β antibody or binding fragment thereof ascompared when not used concurrently with an anti-IL-1β antibody orbinding fragment thereof.

The terms “acute uveitis exacerbation” as used herein refers to anoccurrence of one or more clinical symptoms or manifestations (e.g.,parameters) of uveitis in a subject. Alternatively or in addition, thesubject may be a subject (e.g., at risk subject) that has previouslyexperienced one or more clinical symptoms or manifestations (e.g.,parameters) of uveitis, with an intervening period of clinicallysignificant improvement in one or more uveitis symptoms (e.g., decreaseand/or clinical control of symptoms, symptom free interval). Forexample, the acute uveitis exacerbation may be a first uveitisexacerbation or may be a re-occurrence of a uveitis exacerbation. Theone or more uveitis symptoms during the acute uveitis exacerbation maybe similar to, the same as, or different from (e.g., differentcombination) one or more uveitis symptoms experienced previously. Auveitis flare may be considered an acute uveitis exacerbation.

A variety of methods and techniques for detecting the presence of and/orchanges in symptoms, aspects, parameters or characteristics of diseasestates or conditions referred to herein are known and accepted by thoseof skill in the art. Representative examples of uveitis parameters(e.g., symptoms) that may be examined for changes, such as for example,in the methods of treating or preventing uveitis of the presentdisclosure, may include any or all of visual acuity, vitreous haze,anterior chamber cell score, macular edema, laser flare cell count(e.g., flare score), subretinal pooling, epiretinal membrane formation,hypopyon, subretinal neovascularization, optic disc neovascularization,retinal neovascularization, retinal vasculitis, occlusive vasculitis,peripheral vascular sheathing, inflammatory sheathing, branch retinalvein occlusion, vascular leakage (e.g., fundus fluorescein angiographyleakage score, dual fluorescein angiography and indocyanine greenangiography score), optic disc hyperfluorescence, disc margin staining,optic disc leakage, cystic pooling, posterior pole arcades, retinalcapillary nonperfusion, macular ischemia, pinpoint leaks, retinalstaining, iritis, iridocyclitis, anterior cyclitis, pars planitis,posterior cyclitis, focal choroiditis, multifocal choroiditis, diffusechoroiditis, chorioretinitis, retinochoroiditis, retinitis,neuroretinitis, retinal dysfunction and elevated intraocular pressure.

Examination for changes (e.g., improvement) may be made by standardmedically accepted practices known in the art, such as for example,detailed ophthalmological assessment comprising measurements of visualacuity (e.g., BCVA by ETDRS), intraocular pressure, and vitreous haze;evaluation of retinal findings (infiltrates, inflammatory sheathing,hemorrhages/occlusive vasculitis, and branch retinal vein occlusion);biomicroscopy (e.g., slit-lamp biomicroscopy); ophthalmoscopy (e.g.,indirect ophthalmoscopy of the posterior segment followed by fundusphotography), readings of laser flare cell photometry to track changesin visual acuity and other ocular components inflammation); fluoresceinangiography (e.g., fundus fluorescein angiographic examination, dualfluorescein, angiography (FA) and indocyanine green angiography (ICGA)).

The clinical status of a subjects uveitis may be measured using theUveitis Scoring System (BenEzra, et al., Uveitis Scoring System,Springer-Verlag, Berlin, 1991), which separates the assessment ofuveitis into five components: anterior segment, vitreous, fundus, visualacuity, and fluorescein angiography. This system emphasizes theimportance of the first three components in tracking intraocularinflammation. Components are scored separately, not cumulatively, forexample:

-   -   The anterior segment is graded with slit-lamp biomicroscopy.        Both cells and flares are graded from nil to severe.    -   Vitreous haze resulting from inflammation is examined with a        binocular indirect ophthalmoscope, Scores range from nil to        severe depending on the visibility of the posterior pole.    -   The fundus is graded according to a diagram that divides each        eye into quadrants (e.g., four pre-equatorial and four        post-equatorial sections) and examines each section for retinal        vasculitis, chorioretinal lesions, and neovascularization of the        disc.    -   Visual acuity scores are expressed in the decimal versions of        standard fractions using either Snellen or ETDRS testing charts.    -   Fluorescein angiograms help to determine the extent of retinal        inflammation. Diagrammatically dividing the eye into the        quadrants (e.g., four pre- and post-equatorial segments) used        previously for grading the fundus, the BenEzra system assesses        the inflammation of each section according to 11 criteria.

In some embodiments, methods of the present disclosure may provide animprovement in Ben Ezra score, as measured by the Uveitis ScoringSystem. For example, the methods may provide an improvement (e.g.,decrease) in score of 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more.Alternatively or in addition, the methods of the present disclosure mayprovide an improvement in Ben Ezra score from any score greater than 0to a score of 0.

In some embodiments, visual acuity (e.g., best corrected visual acuity,BCVA) may be measured (e.g., graded) using standard Bailey-Lovie logMAR,Snellen or ETDRS testing charts, which provide for objectivemeasurements. For example, the methods of the present disclosure mayprovide at least a 1 line, at least a 2 line, at least a 3 line, atleast a 4 line or at least a 5 line improvement in visual acuity asmeasured by a Snellen chart, test and/or as illustrated in the followingtable.

Visual/Acuity in Different Notations Feet Meters Decimal Jaeger 20/206/6 1.0 J1+ 20/25 6/7.5 0.8 J1 20/30 6/9 0.7 J2 20/40 6/12 0.5 J3 20/606/15 0.4 J6 20/70 6/21 N/A J7 20/80 6/24 N/A N/A 20/100 6/30 0.2 J1020/150 6/45 N/A N/A 20/200 6/60 0.1 J16 20/400 6/120 0.06 N/A

Alternatively of in addition, the methods of the present disclosure mayprovide at least a 1 line (e.g., 5 letter), at least a 2 line (e.g., 10letter), at least a 3 line (e.g., IS letter), at least a 4 line (e.g.,20 letter), or at least a 5 line (e.g., 25 letter) improvement in visualacuity as measured by ETDRS chart (Ferris et al., 1982, Am. J.Ophthalmol., 94:91-6). In some embodiments, if the subject's visualacuity is so poor that the largest chart letters cannot be read whentested at one meter, then the subject's ability to count lingers,detect, hand motion, or have light perception may be evaluated.

Anterior chamber cell findings may be assessed using the SUN WorkingGroup Grading Scheme (Jabs, et al., Am J Ophthalmol. 140:509-16, 2005).In some embodiments, the anterior segment may be assessed bybiomicroscopy (e.g., slit-lamp biomicroscopy) and anterior chamber cellscore may be measured (e.g., scored). In some embodiments, anteriorchamber cells are scored using the SUN Working Group grading scheme, asshown below.

For example, the methods of the present disclosure may provide animprovement in anterior chamber cell score of at least 1 grade (e.g., 3+to 2+, 1+ to 0.5+), at least 2 grades, at least 3 grades or at least 4grades. Alternatively or in addition, the methods of the presentdisclosure may provide an Improvement in anterior chamber cell scorefrom any of grades 1+ through 4+ to grade 0.5+ or better. Alternativelyor in addition, the methods of the present disclosure may provide animprovement In anterior chamber cell score from any of grades 0.5+through 4+ to grade 0.

The SUN Working Group Grading Scheme for Anterior Chamber Cells GradeCells in Field² 0   <1   0.5+ 1-5 1+  6-15 2+ 16-25 3+ 26-50 4+ >50²Field size is a 1 mm by 1 mm slit beam

In some embodiments, laser flare cell count (e.g., flare score), may bemeasured using laser flare-cell photometry, such as for example asdescribed by Ladas et al., (Survey of Ophthalmology, 50: 27-47, 2005).For example, the methods of the present disclosure may provide animprovement (e.g., reduction) in laser flare cell count (e.g., flarescore) of at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or more, as comparedto initial (e.g., pre-treatment) levels. Alternatively or in addition,the methods of the present disclosure may provide an improvement (e.g.,reduction) in laser flare cell count (e.g., flare score), of at least adecrease in score of 10 or more, 29 or more, 30 or more, 40 or more, 50or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more.

In some embodiments, vitreous haze may be measured using standardmethods, such as for example by ophthalmoscopy (e.g., with binocularindirect ophthalmoscope), with grading, for example, according to theNussenblatt classification (Nussenblatt et al., 1985, Ophthalmol.92:467-71) and/or SUN Working Group's adaptation of the National EyeInstitute system, for grading vitreous haze (Jabs et al, 2005, Am. J.Ophthalmol. 140:5009-16). For example, the methods of the presentdisclosure may provide an improvement (e.g., reduction) in vitreous hazeof at least 1 grade (e.g., 3+ to 2+, 1+ to 0.5+), at least 2 grades, atleast 3 grades or at least 4 grades. Alternatively or in addition, themethods of the present disclosure may provide an improvement in vitreoushaze from any of grades 1+ through 4+ to grade 0.5+ or better.Alternatively or in addition, the methods of the present disclosure mayprovide an improvement in vitreous haze from any of grades 0.5+ through4+ to grade 0.

Additional scoring measurements that may optionally be evaluated, suchas for example, dual fluorescein angiography (FA) and indocyanine greenangiography (ICGA), which may include for FA—optic dischyperfluorescence, macular edema, retinal vascular staining and/orleakage, capillary leakage, retinal capillary nonperfusion,neovascularization of the optic disc, neovascularization elsewhere,pinpoint leaks, and retinal staining and/or subretinal pooling, and forICGA—early stromal vessel hyperfluorescence, choroidal vasculitis, darkdots or areas (excluding atrophy), and/or optic disc hyperfluorescence,are defined by the Angiography Scoring for Uveitis Working Group(Tugai-Tutkun et al., Int Ophthalmol 2010 30(5):529-552; Epub ahead ofprint 2008 Sep. 16).

In some embodiments, intraocular pressure may be assessed using standardaccepted medical practices, such as for example by Goldmann Tonometry.Vascular and/or neurological complications also may be followed. Forexample, subjects with organ involvement such as arterial, aneurysms ordeep vein thrombosis, clinical findings may be followed andcontrast-enhanced spiral computer tomography (CT) imaging may beperformed. For subjects with parenchymal neurologic involvement, acomplete neurologic examination, a contrast-enhanced cranial magneticresonance imaging (MRI) and/or a cerebrospinal fluid (CSF) analysis maybe performed.

Alternatively, or in addition, the following may be evaluated:

-   -   Color fundus photography. Color fundus photography is useful in        documenting the presence of posterior-segment pathology. Color        photography can often highlight subtle clinical findings, and it        is especially useful for establishing 8 baseline and detecting        disease progression over time.    -   Fluorescein angiography. Fluorescein angiography (FA) is useful        in evaluating changes such as breakdown in the blood-retinal        barrier, which can lead to CME and papillitis. FA is also useful        in detecting vascular occlusion from vasculitis, which can be        the result of the numerous causes of posterior uveitis and        choroiditis, as well as complications such as retinal or        choroidal neovascularization (CNV).    -   Indocyanine green angiography. Indocyanine green (ICG)        angiography is used mainly as an adjunct to FA to help evaluate        the choroidal vasculature. The most useful information is        obtained in the later phases of the ICG study. Herbort and        colleagues developed in 1997 a standardized protocol for        administration, and interpretation of posterior uveitis using        ICG. Several conditions, such as birdshot retinochoroiditis, are        much more prominent with ICG angiography.    -   Autofluorescence. Autofluorescence (AF) imaging highlights the        presence of lipofusion in the retinal pigment epithelium (RPE).        Since many posterior uveitic conditions, particularly the white        spot syndromes, affect the outer retina-RPE-choriocapillaries        complex, AF can be a particularly useful noninvasive diagnostic        tool. For Instance, the numerous dots and spots of MEWDS are        much easier to appreciate with fundus AF.    -   B-scan ultrasonography. B-scan ultrasonography has been most        useful in the evaluation of intraocular disorders associated        with opacified media. Opacified media can he caused by        intraocular inflammation and its complications, as well as other        conditions, including: but not limited to corneal opacification,        anterior chamber hyphema or hypopyon, posterior synechiae with        miosis, cataract, vitreous hemorrhage, and retinal detachment.        Ultrasound can also be used to evaluate inflammatory        Infiltration of the choroid, as occurs in chronic uveitis        including Vogt-Koyanagi-Harada (VKH) syndrome, sympathetic        ophthalmia, and combined scleral and choroidal thickening from        scleritis. In these situations, ultrasound becomes useful in        evaluating patients prior to instituting therapy or planning        surgery. In the presence of clear media, high-frequency        ultrasound or ultrasound, biomicroscopy (UBM) can be of        additional use, particularly for examination of the region of        the ciliary body and pars plana, which are often involved in        patients with intermediate uveitis and can be difficult to        visualize clinically. UBM may also identify occult foreign        bodies in cases of chronic uveitis occurring after trauma.    -   Optical coherence tomography. Optical coherence tomography (OCT)        is currently one of the most important imaging techniques used        in the study of uveitis, it enables imaging of the optic nerve        head, nerve fiber layer; retina, choroid, and the vitreoretinal        interface in a noncontact and noninvasive manner, it can be        repeated as often as necessary since there are no serious side        effects in OCT testing. OCT can be used to quantify macular        thickening and thus is an excellent way of diagnosing CME and        monitoring the effectiveness of treatment. OCT can detect        vitreoretinal interface disorders such as epiretinal membranes,        macular holes, and vitreomacular traction, which can assist in        management. OCT is also valuable in the study of the different        types of retinal detachment and the role, location, and density        of an associated exudate. The most important limitation of OCT        is its reliance on relatively clear media for useful images. A        second factor limiting OCT's utility is the need for patient        cooperation with fixation and control of eye movements. These        limitations may prove difficult for photophobic subjects.

In some embodiments, methods of the present disclosure may provide animprovement Ben Ezra score, as measured by the Uveitis Scoring System(BenEzra, et al., Uveitis Scoring System, Springer-Verlag, Berlin,1991). For example, the methods may provide an improvement (e.g.,decrease) in score of 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more.Alternatively or in addition, the methods of the present disclosure mayprovide an improvement in Ben Ezra score from any score greater than 0to a score of 0.

Alternatively or in addition, assessments of other Behçet's diseaseparameters also may be performed.

Alternatively or in addition, assessments may include measures ofbiologic and clinical activity, including non-ocular parameters, such asfor example:

-   -   Inflammatory markers (both CRP and ESR)    -   Analysis of cytokines (e.g., markers for cytokines, such as        inflammatory cytokines), including, but not limited to,        adiponectin, resistin, leptin, visfatin, PAI-1, TNFα, IFNγ,        IL-1, IL-1Ra, IL-6, IL-8, RANTES, IL-1α and MCP-1.

In addition, methods of the present disclosure may provide animprovement (e.g., decrease) in C-reactive protein (CRP) levels. Thereduction in CRP levels is readily measured using standard assays (e.g.,high-sensitivity CRP, ultra-sensitive CRP). As provided by the methodsdisclosed herein, the decrease in C-reactive protein levels may, forexample, be a decrease of ≧0.2, ≧0.4, ≧0.6, ≧0.8, ≧1.0, ≧1.4, ≧1.8,≧2.2, ≧2.6, ≧3.0 mg/L from pre-treatment levels. Alternatively, thedecrease in C-reactive protein levels may, for example, be a decreaseof >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95% frompre-treatment levels.

Further, the methods of the present disclosure may provide animprovement in one or more aspects of quality of life, such as forexample as determined by a quality of life (QOL) assessment (e.g.,SF-36V2 Health Survey, ophthalmological QOL questionnaire).

In some embodiments, methods of treating or preventing a disease orcondition in accordance with the present disclosure may use apre-determined or “routine” schedule for administration of the antibodyor fragment. As used herein a routine schedule refers to a predetermineddesignated period of time between dose administrations. The routineschedule may encompass periods of time which are identical or whichdiffer in length, as long as the schedule is predetermined. Anyparticular combination would be covered by the routine schedule as longas it is determined ahead of time that the appropriate schedule involvesadministration on a certain day.

EXAMPLES

The following examples are intended merely to further illustrate thepractice of the present invention, but should not be construed as in anyway limiting its scope. The disclosures of ail patent and scientificliteratures cited within are hereby expressly incorporated in theirentirety by reference.

Example 1 Clinical Study to Evaluate Treatment of Refractory Uveitiswith an IL-1β Antibody

A clinical trial was undertaken to evaluate treatment with an IL-1βantibody in human subjects diagnosed with uveitis (e.g., acute uveitisexacerbation). More specifically, an open label clinical study wasperformed to examine the safety, PK and clinical activity of a highaffinity IL-1β antibody in subjects with uveitis refractory (e.g.,resistant) to one or more standard of care medications, such as forexample, non-steroid immunosuppressants (e.g., azathioprine, colchicine,cyclosporine, mycophenolate) and/or steroids (e.g., prednisolone).Multiple uveitis parameters were measured in this clinical study.

Subjects enrolled in the clinical trial included those with a uveitisexacerbation having at least acute posterior or panuveitis or retinalvasculitis, and who were also diagnosed with Behçet's disease fulfillingthe international Study Group Classification Criteria. Additionalcriteria for entry into the trial also included:

-   -   Resistant to azathioprine, colchicine, and/or cyclosporine        treatment    -   Receiving stable doses of azathioprine (2 mg/kg) and/or a stable        cyclosporine treatment regimen for at least 1.4 days prior to        Day 0    -   Agreement to discontinue any azathioprine, colchicine, and/or        cyclosporine treatment on Day 0 and for duration of study    -   Subjects were excluded from this study if they met any of the        following criteria:    -   Severe uveitis and a potential visual acuity of less than 0.1        according to Snellen/ETDRS standards for visual acuity    -   Subject has cataract and an assessment of the posterior segment        of his or her uvea was poor or impossible    -   Use of the following medications:        -   NSAID therapy (other than aspirin≦100 mg/day) from 28 days            prior to Day 0 through the end of the study        -   Steroids>10 mg/day from 28 days prior to Day 0 through the            end of the study, with subsequent protocol amendment to            allow participation of one subject receiving prednisolone at            20 mg/day        -   interferon from 28 days prior to Day 0 through the end of            the study

The primary outcome measure was the severity of a subject's uveitis atDays 0 and 28, including degree of improvement in visual acuity andother ocular components (e.g., measured using the Ben-Ezra uveitisscoring system and laser flare meter readings), with periodic follow-upassessments through Day 98.

The prior treatment profiles of seven patients enrolled in thisopen-label study are shown in FIG. 1. All subjects had received priortreatment with azathioprine (AZA) and steroid, and additionally, somesubjects had received prior treatment with colchicine and/orcyclosporine (CysA). Dose amounts for steroid treatment withprednisolone or methylprednisolone are converted to the prednisolone(PRD in FIG. 1) equivalent (e.g., 8 mg methylprednisolone=10 mgprednisolone; 16 mg methylprednisolone=20 mg prednisolone) in theFigure. Subjects continued steroid treatment through the study at theindicated prednisolone dose amounts unless a bolus administration wasrequired at a later time point (post-28 day) as rescue medication.

A single dose of the IL-1β antibody AB7 was administered intravenouslyto the enrolled subjects at a dose of 0.3 mg/kg. Subjects underwentperiodic follow-up assessment. Safety was assessed by pre- andpost-treatment serial measurements of vital signs, clinical laboratoryassessments, and the recording of adverse clinical events. PK data wascollected and analyzed. Changes in the clinical status of the uveitiswere evaluated to gauge long-term and average disease control, using anapproach similar to Sfikakis et al. (Lancet 28:358, 2001). Because thestudy included subjects whose uveitis symptoms have not alreadyresponded to azathioprine, cyclosporins, and/or colchicine (e.g.,treatment refractory), and who suspend these treatments during thecourse of the study, a rapid improvement in subject eye disease in thecourse of this trial would be attributable to study drug.

The primary outcome measure was the progress of uveitis from Day 0 toDay 28, with periodic follow-up assessments through Day 98. The clinicalassessment of uveitis was assessed at every clinic visit (e.g. days 0,1, 4, 7, 14, 21, 28, 56, 98) by standard medically accepted practicesknown in the art, such as for example, detailed ophthalmologicalassessment comprising measurements of visual acuity, intraocularpressure, and vitreous haze; an evaluation of retinal findings(infiltrates, inflammatory sheathing, hemorrhages/occlusive vasculitis,and branch retinal vein occlusion); slit-lamp biomicroscopy; andindirect ophthalmoscopy of the posterior segment followed by fundusphotography. Readings of the laser flare cell photometry was recorded totrack changes in visual acuity and other ocular components. At Days 0(pre-dose) and Day 98 only, a fundus fluorescein angiographicexamination also was performed.

The clinical status of a subject's uveitis was measured using theUveitis Scoring System (BenEzra, et al., Uveitis Scoring System,Springer-Verlag, Berlin, 1991). The BenEzra uveitis scoring systemseparates the assessment of uveitis into five components as follows:anterior segment, vitreous, fundus, visual acuity, and fluoresceinangiography. The system emphasizes the importance of the first threecomponents in tracking intraocular inflammation. Components are scoredseparately, not cumulatively.

-   -   The anterior segment is graded with slit-lamp biomicroscopy.        Both ceils and flares are graded from nil to severe.    -   Vitreous haze resulting from inflammation is examined with a        binocular indirect ophthalmoscope. Scores range from nil to        severe depending on the visibility of the posterior pole.    -   The fundus is graded according to a diagram that divides each        eye into four sections and examines each section for retinal        vasculitis, chorioretinal lesions, and neovascularization.    -   Visual acuity scores are expressed in the decimal versions of        standard fractions using either Snellen or ETDRS testing charts.    -   Fluorescein angiograms help to determine the extent of retinal        inflammation. Diagrammatically dividing the eye into the four        segments used previously for grading the fundus, the BenEzra        system assesses the inflammation of each section according to 11        criteria.

In addition, anterior chamber cell findings were assessed using the SUNWorking Group Grading Scheme (Jabs, et al., Am J Ophthalmol 140:509-16,2005), as follows.

The SUN Working Group Grading Scheme for Anterior Chamber Cells GradeCells in Field² 0   <1   0.5+ 1-5 1+  6-15 2+ 16-25 3+ 26-50 4+ >50²Field size is a 1 mm by 1 mm slit beam

Additional scoring measurements, such as with fluorescein angiography(FA) and indocyanine green angiography (ICGA) are defined by theAngiography Scoring for Uveitis Working Group (Tugal-Tutkun et al., IntOphthalmol, 2010 30(5):539-352, Epub ahead of print 2008 Sep. 16). Inappropriate subjects, the clinical protocol also provided for assessmentof the following of vascular and/or neurological complications. Forsubjects with organ involvement such as arterial aneurysms or deep veinthrombosis, the protocol provided for clinical findings to be followedduring each visit and contrast-enhanced spiral computer tomography (CT)imaging were performed at Day 0 (baseline) and Day 98. For subjects withparenchymal neurologic involvement, the protocol provided for a completeneurologic examination to be performed during each visit, and acontrast-enhanced cranial magnetic resonance imaging (MRI) and acerebrospinal fluid (CSF) analysis to be performed at Day 0 (baseline),Day 28, and Day 98. Additional assessments included measures of biologicand clinical activity, including non-ocular parameters, such as forexample:

-   -   Inflammatory markers (both CRP and ESR)    -   Analysis of cytokines, including, but not limited to,        adiponectin, resistin, leptin, visfatin, PAI-1, TNFα, IFN-γ,        IL-1, IL-1Ra, IL-6, IL-8, RANTES, IL-1α and MCP-1.

Additional assessments of other Behçet's disease parameters (e.g.,non-ocular) also may be performed.

Example 2 Treatment of Refractory Uveitis with an IL-1β Antibody

Data obtained from the study described in Example 1 demonstrated aclinical benefit in the treatment refractory uveitis of all subjects,resulting from administration of the IL-1β antibody, with improvement inmultiple parameters. FIGS. 2-8 show individual subject data, through theDay 98 time points, for the parameters of visual acuity, anteriorchamber cell score, flare score, vitreous haze and Ben Ezra score. FIGS.9 and 10 include images showing specific examples of resolution of ahypopyon and resolution of vitreous haze following treatment with theIL-1β antibody. Overall, in these subjects, findings of intraocularinflammation were resolved in all patients starting from Day 1 followinginfusion, and resolution of retinal findings and vitreous haze wasachieved in 7-21 days.

Example 3 Treatment of Refractory Uveitis with an IL-1β Antibody

A follow up study is undertaken in subjects who were previously enrolledand treated In the study described in Examples 1 and 2. The precedingstudy included subjects whose uveitis symptoms had not already respondedto one or more standard of care medications, including for example,azathioprine, cyclosporine, and/or colchicine (e.g., treatmentrefractory, treatment resistant), and these medications were excludedduring the course of that study. In the study described in this Example,one or more standard of care medications, including for example,non-steroid immunosuppressants (e.g., azathioprine, cyclosporine,colchicine) and/or steroids (e.g., prednisone), are permittedconcurrently with anti-IL-1β antibody treatment.

These subjects may receive additional administrations of an IL-1βantibody over an extended period (e.g., to evaluate the effect onpreventing recurrence of an acute uveitis exacerbation). Morespecifically, an IL-1β antibody is administered to subjects on Days 0,14, and 28, followed by dosing every 4 weeks for up to 2 years.Initially, all subjects receive an intravenous 0.3 mg/kg dose of AB7 onDay 0 and a subcutaneous 0.3 mg/kg dose on Days 14, 21, 56, and 84.After these first five doses, subjects who are stable will have theirdose lowered (e.g., to 0.2 mg/kg) every four weeks for the next fourdoses. Subjects who remain stable after these four doses (e.g., at 0.2mg/kg) will have their dose lowered again (e.g., to 0.1 mg/kg) foradditional administration of antibody every four weeks.

If a subject experiences an acute exacerbation of uveitis (e.g., flare)or an Inadequate response to treatment after having received a dose ateither of the lower dose levels (e.g., 0.1 or 0.2 mg/kg), steroids, suchas prednisolone (oral or IV) may be administered as a bolus plus taper.In addition, the subject's next scheduled dose of IL-1β. antibody may beincreased to either of the previous dose levels. Following such anincrease in dose level, if the subject remains stable through four dosesat the higher dose level, the dose level may again be reduced.

Subjects undergo periodic follow-up assessments for up to 111 weeks.Safety is assessed by pre- and post-treatment serial measurements ofvital signs, clinical laboratory assessments, and the recording ofadverse clinical events. PK data is collected and analyzed. Clinicalprogress of uveitis and any vascular/neurological complications areassessed as described previously. Changes in the clinical status of theuveitis in subjects diagnosed with Behçet's disease are evaluated togauge long term and average disease control, using an approach similarto that used by Sfikakis et al. (Lancet 28:358, 2001). The primaryoutcome measure is the progress or stability of uveitis (e.g.,preventing recurrence of acute uveitis exacerbation) during the 2 yearsof this extension study. The clinical assessment of uveitis (assessed atevery clinic visit) includes a detailed ophthalmological assessmentcomprising measurements of visual acuity, and vitreous haze; anevaluation of fundus findings (infiltrates, inflammatory sheathing,hemorrhages/occlusive vasculitis, and branch retinal vein occlusion);slit-lamp biomicroscopy of the anterior segment; and indirectophthalmoscopy of the posterior segment followed by fundus photography.Readings of the laser flare cell photometry are recorded to trackchanges in visual acuity and other ocular components. On Days 0, 112,224, 330, 448, 560, 672, and 756, the ophthalmological assessmentincludes a fundus fluorescein angiographic examination to evaluate theextent of retinal inflammation. The clinical status of a subject'suveitis is measured and scored using the methods described previously.

In appropriate subjects, vascular and/or neurological complications arefollowed. For subjects with organ involvement such as arterial aneurysmsor deep vein thrombosis, clinical findings are followed during, eachvisit and contrast-enhanced spiral CT imaging is performed as clinicallyindicated. For subjects with parenchymal neurologic involvement, acomplete neurologic examination is performed during each visit, and acontrast-enhanced cranial MRI and a cerebrospinal fluid (CSF) analysisis performed as clinically indicated. Inflammatory markers (CRP, ESR,and cytokines) are collected as additional measures of the biologicalactivity of the antibody.

Example 4 Inhibiting an Acute Uveitis Exacerbation with an IL-1βAntibody

Additional clinical trials may be performed, and may include forexample, the same or alternative dosages and dosing regimens, longertreatment and/or observation periods, greater numbers of subjects pergroup, subjects (e.g., at risk subjects) not currently suffering fromacute uveitis attack (e.g., acute uveitis exacerbation) but with priorhistory of one or more uveitis attacks (e.g., within previous 6 months,12 months), subjects continuing treatment with standard of care ortapering doses of one or more additional active agents (e.g.,non-steroid immunosuppressant, non-steroid anti-inflammatory,azathioprine, steroid) and subjects diagnosed with alternative forms ofuveitis (e.g., non-infectious, infectious) and/or with one or more otherdiseases or conditions (e.g., Inflammatory diseases).

For example, a clinical trial is performed in patients (e.g., Behçet'sdisease patients) who have been diagnosed with a history of recurrentuveitis (e.g., refractory uveitis, resistant uveitis), and thus are atrisk for additional uveitis exacerbations, in order to assess thetherapeutic effect of treatment with an IL-1β antibody to inhibit anacute uveitis exacerbation (e.g., uveitis flare, severe exacerbation).Subjects (e.g., at risk subjects) with a history of recurrent uveitis(e.g., uveitis flare/exacerbation within 8 months, uveitisflare/exacerbation within 12 months, uveitis flare/exacerbation within18 months), but not experiencing a current acute uveitis exacerbation(e.g., within previous 1 month, within previous 3 months) and who arecurrently stable on one or more standard of care medications, includingfor example, a non-steroid immunosuppressant (e.g., azathioprine,cyclosporine, mycophenolate, methotrexate) and/or steroids (e.g.,prednisone, prednisolone), are enrolled in this clinical study.

Groups of subjects (e.g., ten subjects, twenty-five subjects, fifty ormore subjects) continue to receive standard of care treatment andadditionally receive monthly treatment with an IL-1β antibody (e.g.,0.03 mg/kg dose, 0.3 mg/kg dose, 1.0 mg/kg dose, 30 mg dose, 100 mgdose) or placebo via subcutaneous injection. Optionally, the subject mayreceive an initial dose of antibody that is higher (e.g., 2-fold more)and/or delivered by an alternative route (e.g., IV) compared tosubsequent doses of antibody. Subjects are then monitored, for exampleas described herein (e.g., in previous examples), for an acuteexacerbation of uveitis during the treatment period (e.g., 6 monthstreatment period, 12 months treatment period). An acute uveitisexacerbation “event” may include not only deterioration in one or moreconditions (e.g., decrease in visual acuity, increase in vitreous haze,retinal infiltrates or vasculitis), but also initiation of one or morerescue medications (e.g., bolus steroid treatment, new immunosuppressivetherapy).

Therapeutic effect of the IL-1β antibody treatment is determined bycomparing the antibody and placebo treatment groups for number ofsubjects In the group experiencing an acute exacerbation of uveitis(e.g. within a specified time period) and/or the time to acute uveitisexacerbation.

Example 5 Treatment of Uveitis with an IL-1β Antibody Tapering ofStandard of Care Treatment with Pharmaceutical Composition

A clinical trial is performed in patients (e.g., Behçet's diseasepatients) who have been diagnosed with a history of recurrent uveitis(e.g., refractory uveitis, resistant uveitis), in order to assess thetherapeutic effect of treatment with an IL-1β antibody, and the abilityof the antibody to inhibit an acute uveitis exacerbation (e.g., uveitisflare). Subjects with a history of recurrent uveitis, currentlyreceiving one or more standard of care medications, including forexample, a non-steroid immunosuppressant (e.g., azathioprine) and/or asteroid (e.g., prednisolone), and who are experiencing an acute uveitisexacerbation, are enrolled in this clinical study. The enrollmentcriteria for an acute uveitis exacerbation may include any of theaforementioned parameters, such as for example vitreous haze (e.g., ≧1+,≧2+), decrease in visual acuity attributed to the exacerbation (e.g., nobetter than 20/40 ETDRS BCVA, ≧15-letter ETDRS or 2-line Snellendecrease), or retinal infiltrates and/or retinal vasculitis.

All subjects continue to receive standard of care treatment andadditionally Initiate treatment with an IL-1β antibody (e.g., 0.03 mg/kgdose, 0.3 mg/kg dose, 1.0 mg/kg dose, 30 mg dose, 100 mg dose) viaintravenous or subcutaneous administration to resolve the acuteexacerbation. A second, optional dose of antibody may be administered(e.g., intravenous, subcutaneous) at 14 days. Patients are monitoredweekly for response to the IL-1β antibody treatment and at apredetermined time following a response (e.g., 1 week later), generallyby day 28, all subjects that have responded to treatment are randomizedinto one of two groups, with the first group continuing to receivestandard of care treatment plus the IL-1β antibody (e.g., monthly) andthe second group continuing to receive standard of care treatment andadditionally placebo. Optionally, the subject may receive the initialdose of antibody in an amount that is higher (e.g., 2-fold more) and/ordelivered by an alternative route (e.g., IV) compared to subsequentdoses of antibody.

Subjects (e.g., at risk subjects) are then monitored as described herein(e.g., in previous examples) for recurrence of an additional acuteexacerbation of uveitis during the second phase of the treatment period(e.g., 6 months treatment period, 12 months treatment period). An acuteuveitis exacerbation “event” may include not only deterioration in oneor more conditions (e.g., decrease in visual acuity, increase invitreous haze, retinal infiltrates or retinal vasculitis), but alsoinitiation of one or more rescue medications (e.g., bolus steroidtreatment, new immunosuppressive therapy). In addition, the dose levelof one or more standard of care drugs, such as for example animmunosuppressant (e.g., azathioprine, cyclosporine, mycophenolate)and/or steroid (e.g., prednisone), may optionally be decreased ortapered during the treatment period. Therapeutic effect of the IL-1βantibody treatment in preventing further acute uveitis exacerbations isdetermined by comparing the antibody and placebo treatment groups fornumber of subjects In the group experiencing an acute exacerbation ofuveitis (e.g., within a specified time period, upon a specified numberof subjects in a group experiencing an exacerbation) and/or the time toacute uveitis exacerbation.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise Indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Wherever an open-ended term isused to describe a feature or element of the invention, it isspecifically contemplated that a closed-ended term can be used in placeof the open-ended term without departing from the spirit and scope ofthe invention. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Molanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseworking in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law,. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A method of inhibiting uveitis in a subject, the method comprisingadministering to the subject an effective amount of anti-IL-1β antibodyor binding fragment thereof.
 2. The method of claim 1, whereininhibiting uveitis in a subject is inhibiting an acute uveitisexacerbation.
 3. The method of claim 1, wherein said method ofinhibiting uveitis in a subject is a method of preventing uveitis in thesubject.
 4. The method of claim 1, wherein said method of inhibitinguveitis in a subject is a method of treating uveitis in the subject. 5.The method of claim 1, wherein said method of inhibiting uveitis in asubject increases in the interval between acute uveitis exacerbations.6. The method of claim 1, wherein said method of inhibiting uveitis in asubject decreases the frequency of acute uveitis exacerbations.
 7. Themethod of claim 1, wherein said method of inhibiting uveitis in asubject decreases the likelihood of experiencing an acute uveitisexacerbation.
 8. The method of claim 1, wherein said method ofinhibiting uveitis in a subject prevents an acute uveitis exacerbation.9. The method of claim 1, wherein said method of inhibiting uveitis in asubject decreases the severity of an acute uveitis exacerbation.
 10. Themethod of claim 1, wherein the treatment refractory uveitis is uveitisthat is refractory to treatment with a pharmaceutical compositioncomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid.
 11. The method of claim 10, wherein thenon-steroid immunosuppressant is a DNA synthesis inhibitor, acyclosporine, a mycophenolate or a colchicine.
 12. The method of claim11, wherein the DNA synthesis inhibitor is azathioprine, an alkylatingagent, an anti-metabolite, X-ray therapy, chlorambucil orcyclophosphamide.
 13. The method of claim 10, wherein the non-steroidanti-inflammatory is a TNF inhibitor, an IL-6 inhibitor or an IL-17inhibitor.
 14. The method of claim 10, wherein the steroid is a steroidhormone selected from the group consisting of prednisone,methylprenisolone, prednisolone, a cortisol, an andrenocorticotrophichormone and a glucocorticoid.
 15. The method of claim 1, wherein, thesubject is receiving concurrently for the inhibition of said uveitis oneor two pharmaceutical compositions comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid.
 16. Themethod of claim 15, wherein the subject is receiving concurrently forthe inhibition of said uveitis one pharmaceutical composition comprisinga non-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid.
 17. The method of claim 1, wherein the subject is not receivingconcurrently for the inhibition of said uveitis a pharmaceuticalcomposition selected from the group consisting of a pharmaceuticalcomposition comprising a non-steroid immunosuppressant, a pharmaceuticalcomposition comprising a non-steroid anti-inflammatory and apharmaceutical composition comprising a steroid.
 18. The method of claim17, wherein the subject is not receiving concurrently for the inhibitionof said uveitis a pharmaceutical composition comprising a non-steroidimmunosuppressant.
 19. The method of claim 17, wherein the subject isnot receiving concurrently for the inhibition of said uveitis apharmaceutical composition comprising a non-steroid anti-inflammatory.20. The method of claim 1, wherein the subject has received priortreatment for uveitis with one or more pharmaceutical compositionscomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid.
 21. The method of claim 20, wherein thesubject had an adverse reaction or hypersensitivity to said priortreatment of uveitis with one or more pharmaceutical compositionscomprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid.
 22. The method of claim 20, wherein thesubject failed said prior treatment of uveitis with one or morepharmaceutical compositions comprising a non-steroid immunosuppressant,a non-steroid anti-inflammatory or a steroid.
 23. The method of claim 1,wherein, the subject is receiving concurrently for the inhibition ofsaid uveitis at least one pharmaceutical composition comprising anon-steroid immunosuppressant, a non-steroid anti-inflammatory or asteroid, and wherein said method provides a reduction in the dosage ofsaid at least one pharmaceutical composition.
 24. The method of claim23, wherein said reduction in the dosage is a reduction in the dose ofsaid at least one pharmaceutical composition, as compared to the doseprior to administering the anti-IL-1β antibody or binding fragmentthereof.
 25. The method of claim 23, wherein said reduction in thedosage is a reduction in the frequency of doses of said at least onepharmaceutical composition, as compared to the frequency of doses priorto administering the anti-IL-1β antibody or binding fragment thereof.26. The method of claim 23, wherein the dosage of a pharmaceuticalcomposition comprising a non-steroid immunosuppressant is reduced. 27.The method of claim 26, wherein, the non-steroid immunosuppressant is aDNA synthesis inhibitor, a cyclosporine, mycophenolate or a colchicine.28. The method of claim 27, wherein the DNA synthesis inhibitor isazathioprine or methotrexate.
 29. The method of claim 23, wherein thedosage of a pharmaceutical composition comprising a steroid is reduced.30. The method of claim 29, wherein the steroid is a steroid hormoneselected from the group consisting of prednisolone, a cortisol, anandrenocorticotrophic hormone and a glucocorticoid.
 31. A method oftreating uveitis in a subject, the method comprising administering tothe subject an effective amount of anti-IL-1β antibody or bindingfragment thereof.
 32. The method of claim 31, wherein the treatmentrefractory uveitis is uveitis that is refractory to treatment with apharmaceutical composition comprising a non-steroid immunosuppressant, anon-steroid anti-inflammatory or a steroid.
 33. A method of treating orpreventing uveitis in a subject, the method comprising administering tothe subject an effective amount of anti-IL-1β antibody or bindingfragment thereof, wherein the subject is receiving concurrently for thetreatment or prevention of said uveitis one or two pharmaceuticalcompositions comprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid.
 34. The method of claim 33, wherein thesubject is receiving concurrently for the treatment or prevention ofsaid uveitis one pharmaceutical composition comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid.
 35. Amethod of treating or preventing uveitis in a subject, the methodcomprising administering to the subject an effective amount ofanti-IL-1β antibody or binding fragment thereof, wherein the subject isnot receiving concurrently for the treatment or prevention of saiduveitis a pharmaceutical composition selected from the group consistingof a pharmaceutical composition comprising a non-steroidimmunosuppressant, a pharmaceutical composition comprising a non-steroidanti-inflammatory and a pharmaceutical composition comprising a steroid.36-37. (canceled)
 38. A method of inhibiting an acute uveitisexacerbation in a subject, the method comprising administering to thesubject an effective amount of anti-IL-1β antibody or binding fragmentthereof, wherein the subject has received prior treatment for uveitiswith one or more pharmaceutical compositions comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid.
 39. Themethod of claim 38, wherein the subject had an adverse reaction orhypersensitivity to, or failed said prior treatment of uveitis with oneor more pharmaceutical compositions comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid. 40.(canceled)
 41. A method of inhibiting an acute uveitis exacerbation in asubject, the method comprising administering to the subject an effectiveamount of anti-IL-1β antibody or binding fragment thereof, wherein thesubject is receiving concurrent treatment for said uveitis with one ortwo pharmaceutical compositions comprising a non-steroidimmunosuppressant, a non-steroid anti-inflammatory or a steroid.
 42. Themethod of claim 38, wherein said inhibiting an acute uveitisexacerbation is an increase in the interval between acute uveitisexacerbations, a decrease in the frequency of acute uveitisexacerbations, a decrease in the likelihood of experiencing an acuteuveitis exacerbation, preventing an acute uveitis exacerbation ordecreasing the severity of an acute uveitis exacerbation.
 43. The methodof claim 41, wherein said inhibiting an acute uveitis exacerbation is anincrease in the interval between acute uveitis exacerbations, a decreasein the frequency of acute uveitis exacerbations, a decrease in thelikelihood of experiencing an acute uveitis exacerbation, preventing anacute uveitis exacerbation or decreasing the severity of an acuteuveitis exacerbation. 44-46. (canceled)
 47. A method of treating orpreventing uveitis in a subject, the method comprising administering tothe subject an effective amount of anti-IL-1β antibody or bindingfragment thereof, wherein the subject is receiving concurrently for thetreatment or prevention of said uveitis at least one pharmaceuticalcomposition comprising a non-steroid immunosuppressant, a non-steroidanti-inflammatory or a steroid, and wherein said method provides areduction in the dosage of said at least one pharmaceutical composition.48-55. (canceled)
 56. The method of claim 1, wherein said method resultsin an improvement in Ben-Ezra uveitis score.
 57. The method of claim 1,wherein said method results in an improvement in anterior uveitis orposterior uveitis.
 58. The method of claim 1, wherein said methodresults in an improvement in at least one or two parameters selectedfrom visual acuity, vitreous haze, anterior chamber cell score, macularedema, laser flare cell count, subretinal pooling, epiretinal membraneformation, hypopyon, subretinal neovascularization, optic discneovascularization, retinal neovascularization, retinal infiltrates,retinal vasculitis, occlusive vasculitis, peripheral vascular sheathing,inflammatory sheathing, branch retinal vein occlusion, fundusfluorescein angiography leakage score, optic disc hyperfluorescence,disc margin staining, optic disc leakage, cystic pooling, posterior polearcades, retinal capillary nonperfusion, macular ischemia, pinpointleaks, retinal staining, iritis, iridocyclitis, anterior cyclitis, parsplanitis, posterior cyclitis, focal choroiditis, multifocal choroiditis,diffuse choroiditis, chorioretinitis, retinochoroiditis, retinitis,neuroretinitis, retinal dysfunction and elevated intraocular pressure.59-61. (canceled)
 62. The method of claim 58, wherein said methodresults in an improvement in at least two parameters selected fromvisual acuity, vitreous haze, laser flare-cell count and retinalvasculitis.
 63. (canceled)
 64. A method of treating uveitis in asubject, the method comprising: 1) diagnosing uveitis in the subject,and 2) administering to the subject of step 1) an effective amount ofanti-IL-1β antibody or binding fragment thereof, wherein said methodresults in an improvement in anterior uveitis or posterior uveitis. 65.The method of claim 64, wherein the subject diagnosed with uveitis is asubject diagnosed with panuveitis.
 66. The method of claim 64, whereinsaid method further results in an improvement in intermediate uveitis.67. A method of treating uveitis in a subject, the method comprising: 1)diagnosing uveitis in the subject, and 2) administering to the subjectof step 1) an effective amount of anti-IL-1β antibody or bindingfragment thereof, wherein said method results in an improvement in atleast one or two parameters selected from visual acuity, vitreous haze,anterior chamber cell score, macular edema, laser flare cell count,subretinal pooling, epiretinal membrane formation, hypopyon, subretinalneovascularization, optic disc neovascularization, retinalneovascularization, retinal infiltrates, retinal vasculitis, occlusivevasculitis, peripheral vascular sheathing, inflammatory sheathing,branch retinal vein occlusion, fundus fluorescein angiography leakagescore, optic disc hyperfluorescence, disc margin staining, optic discleakage, cystic pooling, posterior pole arcades, retinal capillarynonperfusion, macular ischemia, pinpoint leaks, retinal staining,iritis, iridocyclitis, anterior cyclitis, pars planitis, posteriorcyclitis, focal choroiditis, multifocal choroiditis, diffusechoroiditis, chorioretinitis, retinochoroiditis, retinitis,neuroretinitis, retinal dysfunction and elevated intraocular pressure.68-70. (canceled)
 71. The method of claim 67, wherein said methodresults in an improvement in at least two parameters selected fromvisual acuity, vitreous haze, laser flare-cell count and retinalvasculitis.
 72. The method of claim 64, wherein the method results in animprovement in Ben Ezra score.
 73. The method of claim 1, wherein theuveitis in non-infectious uveitis.
 74. The method of claim 1, whereinthe subject has been diagnosed with a disease or condition selected fromBehçet's disease, spondyloarthritides, psoriatic arthritis, psoriasis,inflammatory bowel disease, ulcerative colitis, sarcoidosis,tubulointerstitial nephritis and uveitis (TINU) syndrome, rheumatoidarthritis, Kawasaki disease, Sjögren's syndrome, systemic lupuserythematosus, polyarteritis, Reiter disease, Wegener's granulomatosis,Vogt-Koyanagi-Harada syndrome, systemic juvenile idiopathic arthritisand granulomatous angiitis.
 75. The method of claim 1, wherein thesubject has been diagnosed with cytomegalovirus infection,toxoplasmosis, syphilis, tuberculosis, cat scratch disease, Lymedisease, West Nile virus infection, herpes simplex virus infection,human immunodeficiency virus infection, fungal infection orvaricella-zoster infection.
 76. The method of claim 1, wherein thesubject has been diagnosed with a disease or condition selected frompars planitis, multiple sclerosis, sympathetic ophthalmia, birdshotchoroidopathy, immune recovery uveitis, lymphoma and idiopathic uveitis.77. The method of claim 1, wherein the antibody or antibody fragmentbinds to human IL-1β with a dissociation constant of about 1 nM or less,about 250 pM or less, about 50 pM or less, about 10 pM or less, or about1 pM or less. 78-85. (canceled)
 86. The method of claim 1, wherein theantibody or fragment thereof competes with the binding of an antibodyhaving the light chain variable region of SEQ ID NO:5 and the heavychain variable region of SEQ ID NO:6.
 87. The method of claim 1, whereinthe antibody or fragment thereof binds to an epitope of IL-1β that issubstantially the same as the epitope bound by an antibody having thelight chain variable region of SEQ ID NO:5 and the heavy chain variableregion of SEQ ID NO:6.
 88. The method of claim 1, wherein the antibodyor antibody fragment binds to an epitope incorporating Glu64 of IL-1β.89. The method of claim 1, wherein the antibody or antibody fragmentbinds to amino acids 1-34 of the N terminus of IL-1β. 90-91. (canceled)92. The method of claim 1, wherein the antibody or antibody fragment isadministered in one or more doses of about 3 mg/kg or less, about 1mg/kg or less, about 0.3 mg/kg or less, about 0.1 mg/kg or less, about0.03 mg/kg or less, or about 0.01 mg/kg or less of antibody or fragment.93-97. (canceled)
 98. The method of claim 92, wherein the one or moredoses are at least 0.01 mg/kg of antibody or fragment.
 99. The method ofclaim 1, wherein the antibody or fragment is administered as a fixeddose, independent of a dose per subject weight ratio.
 100. The method ofclaim 99, wherein the antibody or fragment is administered in one ormore doses of 500 mg or less, 250 mg or less, 100 mg or less, 25 mg orless, 10 mg or less, or 0.1 mg or less of antibody or fragment. 101-105.(canceled)
 106. The method of claim 99, wherein the antibody or fragmentis administered in one or more doses of at least 1.0 mg of antibody orfragment.
 107. (canceled)
 108. The method of claim 1, wherein theanti-IL-1β antibody or fragment is administered by subcutaneous,intravenous, intraocular or intramuscular injection
 109. (canceled) 110.The method of claim 1, wherein administration of an initial dose of theantibody or antibody fragment is followed by the administration of oneor more subsequent doses, and wherein said one or more subsequent dosesare in an amount that is approximately the same or less than the initialdose.
 111. The method of claim 1, wherein administration of an initialdose of the antibody or antibody fragment is followed by theadministration of one or more subsequent doses, and wherein at least oneof the subsequent doses is in an amount that is more than the initialdose.
 112. The method of claim 1, wherein the anti-IL-1β antibody orbinding fragment thereof is administered in a dose amount and frequencysufficient to maintain a systemic trough serum concentration of at leastabout 0.5 μg/mL, at least about 1.0 μg/mL, or at least about 2.0 μg/mL.113-115. (canceled)
 116. A method of inhibiting uveitis in a subject,the method comprising administering to the subject an anti-IL-1βantibody or binding fragment thereof in a dose amount and frequencysufficient to maintain a systemic trough serum concentration of at leastabout 0.5 μg/mL, at least about 1.0 μq/mL, or at least about 2.0 μg/mL.117-122. (canceled)